RNA INTERFERENCE MEDIATED INHIBITION OF INTERLEUKIN AND INTERLEUKIN RECEPTOR GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)

ABSTRACT

This invention relates to compounds, compositions, and methods useful for modulating interleukin and/or interleukin receptor gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of interleukin and/or interleukin receptor gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of interleukin and/or interleukin receptor genes such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, and IL-27 genes and IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL- 11 R, IL-12R, IL-13R, IL-14R, IL-15R, IL-16R, IL-17R, IL-18R, IL-19R, IL-20R, IL-21R, 1L-22R, IL-23R, IL-24R, IL-25R, IL-26R, and IL-27R genes.

This application is a continuation of U.S. patent application Ser. No. 10/863,973, filed on Jun. 9, 2004, which is a continuation-in-part of International Patent Application No. PCT/US03/04566, filed Feb. 14, 2003, and parent U.S. patent application Ser. No. 10/863,973 is also a continuation-in-part of International Patent Application No. PCT/US04/16390, filed May 24, 2004, which is a continuation-in-part of U.S. patent application Ser. No. 10/826,966, filed Apr. 16, 2004, which is continuation-in-part of U.S. patent application Ser. No. 10/757,803, filed Jan. 14, 2004, which is a continuation-in-part of U.S. patent application Ser. No. 10/720,448, filed Nov. 24, 2003, which is a continuation-in-part of U.S. patent application Ser. No. 10/693,059, filed Oct. 23, 2003, which is a continuation-in-part of U.S. patent application Ser. No. 10/444,853, filed May 23, 2003, which is a continuation-in-part of International Patent Application No. PCT/US03/05346, filed Feb. 20, 2003, and a continuation-in-part of International Patent Application No. PCT/US03/05028, filed Feb. 20, 2003, both of which claim the benefit of U.S. Provisional Application No. 60/358,580, filed Feb. 20, 2002, U.S. Provisional Application No. 60/363,124, filed Mar. 11, 2002, U.S. Provisional Application No. 60/386,782, filed Jun. 6, 2002, U.S. Provisional Application No. 60/406,784, filed Aug. 29, 2002, U.S. Provisional Application No. 60/408,378, filed Sep. 5, 2002, U.S. Provisional Application No. 60/409,293, filed Sep. 9, 2002, and U.S. Provisional Application No. 60/440,129, filed Jan. 15, 2003. The instant application claims the benefit of all the listed applications, which are hereby incorporated by reference herein in their entireties, including the drawings.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR §1.52(e)(5), is incorporated herein by reference. The sequence listing text file submitted via EFS contains the file “SequenceListing17USCNT”, created on Sep. 3, 2008, which is 483,552 bytes in size.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of interleukin gene expression and/or activity, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, and IL-27 genes and genes encoding interleukin receptors of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, and IL-27 genes. The present invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and/or activity of genes involved in interleukin gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against interleukin gene expression. Such small nucleic acid molecules are useful, for example, in providing compositions for treatment or prevention of traits, diseases and conditions that can respond to modulation of interleukin gene expression in a subject, such as inflammatory, respiratory, pulmonary, autoimmune, cardiovascular, neurodegenerative, and/or proliferative and cancerous diseases, traits, or conditions.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. The discussion is provided only for understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.

RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). The corresponding process in plants (Heifetz et al., International PCT Publication No. WO 99/61631) is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response through a mechanism that has yet to be fully characterized. This mechanism appears to be different from other known mechanisms involving double-stranded RNA-specific ribonucleases, such as the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094; 5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17, 503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101, 235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000, Nature, 404, 293). Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101, 25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283 and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., International PCT Publication No. WO 01/75164, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al., International PCT Publication No. WO 01/75164) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21-nucleotide siRNA duplexes are most active when containing 3′-terminal dinucleotide overhangs. Furthermore, complete substitution of one or both siRNA strands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3′-terminal siRNA overhang nucleotides with 2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end of the guide sequence (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of an siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhanging segments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to four nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated, whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al., International PCT Publication No. WO 01/75164). In addition, Elbashir et al., supra, also report that substitution of siRNA with 2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al., International PCT Publication No. WO 00/44914, and Beach et al., International PCT Publication No. WO 01/68836 preliminarily suggest that siRNA may include modifications to either the phosphate-sugar backbone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom, however, neither application postulates to what extent such modifications would be tolerated in siRNA molecules, nor provides any further guidance or examples of such modified siRNA. Kreutzer et al., Canadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double-stranded RNA-dependent protein kinase PKR, specifically 2′-amino or 2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-C methylene bridge. However, Kreutzer et al. similarly fails to provide examples or guidance as to what extent these modifications would be tolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certain chemical modifications targeting the unc-22 gene in C. elegans using long (>25 nt) siRNA transcripts. The authors describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and observed that RNAs with two phosphorothioate modified bases also had substantial decreases in effectiveness as RNAi. Further, Parrish et al. reported that phosphorothioate modification of more than two residues greatly destabilized the RNAs in vitro such that interference activities could not be assayed. Id. at 1081. The authors also tested certain modifications at the 2′-position of the nucleotide sugar in the long siRNA transcripts and found that substituting deoxynucleotides for ribonucleotides produced a substantial decrease in interference activity, especially in the case of Uridine to Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id. In addition, the authors tested certain base modifications, including substituting, in sense and antisense strands of the siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine for guanosine. Whereas 4-thiouracil and 5-bromouracil substitution appeared to be tolerated, Parrish reported that inosine produced a substantial decrease in interference activity when incorporated in either strand. Parrish also reported that incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resulted in a substantial decrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al., International PCT Publication No. WO 01/68836, describes specific methods for attenuating gene expression using endogenously-derived dsRNA. Tuschl et al., International PCT Publication No. WO 01/75164, describe a Drosophila in vitro RNAi system and the use of specific siRNA molecules for certain functional genomic and certain therapeutic applications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubts that RNAi can be used to cure genetic diseases or viral infection due to the danger of activating interferon response. Li et al., International PCT Publication No. WO 00/44914, describe the use of specific long (141 bp-488 bp) enzymatically synthesized or vector expressed dsRNAs for attenuating the expression of certain target genes. Zernicka-Goetz et al., International PCT Publication No. WO 01/36646, describe certain methods for inhibiting the expression of particular genes in mammalian cells using certain long (550 bp-714 bp), enzymatically synthesized or vector expressed dsRNA molecules. Fire et al., International PCT Publication No. WO 99/32619, describe particular methods for introducing certain long dsRNA molecules into cells for use in inhibiting gene expression in nematodes. Plaetinck et al., International PCT Publication No. WO 00/01846, describe certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific long dsRNA molecules. Mello et al., International PCT Publication No. WO 01/29058, describe the identification of specific genes involved in dsRNA-mediated RNAi. Pachuck et al., International PCT Publication No. WO 00/63364, describe certain long (at least 200 nucleotide) dsRNA constructs. Deschamps Depaillette et al., International PCT Publication No. WO 99/07409, describe specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. Waterhouse et al., International PCT Publication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describe certain methods for decreasing the phenotypic expression of a nucleic acid in plant cells using certain dsRNAs. Driscoll et al., International PCT Publication No. WO 01/49844, describe specific DNA expression constructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. For example, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describe specific chemically-modified dsRNA constructs targeting the unc-22 gene of C. elegans. Grossniklaus, International PCT Publication No. WO 01/38551, describes certain methods for regulating polycomb gene expression in plants using certain dsRNAs. Churikov et al., International PCT Publication No. WO 01/42443, describe certain methods for modifying genetic characteristics of an organism using certain dsRNAs. Cogoni et al., International PCT Publication No. WO 01/53475, describe certain methods for isolating a Neurospora silencing gene and uses thereof. Reed et al., International PCT Publication No. WO 01/68836, describe certain methods for gene silencing in plants. Honer et al., International PCT Publication No. WO 01/70944, describe certain methods of drug screening using transgenic nematodes as Parkinson's Disease models using certain dsRNAs. Deak et al., International PCT Publication No. WO 01/72774, describe certain Drosophila-derived gene products that may be related to RNAi in Drosophila. Arndt et al., International PCT Publication No. WO 01/92513 describe certain methods for mediating gene suppression by using factors that enhance RNAi. Tuschl et al., International PCT Publication No. WO 02/44321, describe certain synthetic siRNA constructs. Pachuk et al., International PCT Publication No. WO 00/63364, and Satishchandran et al., International PCT Publication No. WO 01/04313, describe certain methods and compositions for inhibiting the function of certain polynucleotide sequences using certain long (over 250 bp), vector expressed dsRNAs. Echeverri et al., International PCT Publication No. WO 02/38805, describe certain C. elegans genes identified via RNAi. Kreutzer et al., International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP 1144623 B1 describes certain methods for inhibiting gene expression using dsRNA. Graham et al., International PCT Publications Nos. WO 99/49029 and WO 01/70949, and AU 4037501 describe certain vector expressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559, describe certain methods for inhibiting gene expression in vitro using certain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi. Martinez et al., 2002, Cell, 110, 563-574, describe certain single-stranded siRNA constructs, including certain 5′-phosphorylated single-stranded siRNAs that mediate RNA interference in HeLa cells. Harborth et al., 2003, Antisense & Nucleic Acid Drug Development, 13, 83-105, describe certain chemically and structurally modified siRNA molecules. Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically and structurally modified siRNA molecules. Woolf et al., International PCT Publication Nos. WO 03/064626 and WO 03/064625 describe certain chemically modified dsRNA constructs.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods useful for modulating interleukin and/or interleukin receptor gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of interleukin and/or interleukin receptor gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of interleukin and/or interleukin receptor genes.

An siNA of the invention can be unmodified or chemically-modified. An siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. The instant invention also features various chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating interleukin and/or interleukin receptor gene expression or activity in cells by RNA interference (RNAi). The use of chemically-modified siNA improves various properties of native siNA molecules through increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Further, contrary to earlier published studies, siNA having multiple chemical modifications retains its RNAi activity. The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

In one embodiment, the invention features one or more siNA molecules and methods that independently or in combination modulate the expression of interleukin and/or interleukin receptor genes encoding proteins, such as proteins comprising interleukins (e.g., IL-1-IL-27) and interleukin receptors (e.g., IL-IR-1L-27R), such as genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I, referred to herein generally as interleukin and/or interleukin receptor. The description below of the various aspects and embodiments of the invention is provided with reference to exemplary interleukin and interleukin receptor genes referred to herein as interleukin and/or interleukin receptor. However, the various aspects and embodiments are also directed to other interleukin and/or interleukin receptor genes, such as interleukin and/or interleukin receptor homolog genes, transcript variants, and polymorphisms (e.g., single nucleotide polymorphism, (SNPs)) associated with certain interleukin and/or interleukin receptor genes, for example genes associated with diseases, traits, or conditions described herein or otherwise known in the art. As such, the various aspects and embodiments are also directed to other genes that are involved in interleukin and/or interleukin receptor mediated pathways of signal transduction or gene expression. These additional genes can be analyzed for target sites using the methods described for interleukin and/or interleukin receptor genes herein. Thus, the modulation of other genes and the effects of such modulation of the other genes can be performed, determined, and measured as described herein.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a interleukin and/or interleukin receptor gene, wherein said siNA molecule comprises about 19 to about 21 base pairs.

In one embodiment, the invention features an siNA molecule that down-regulates expression of a interleukin and/or interleukin receptor gene, for example, wherein the interleukin and/or interleukin receptor gene comprises interleukin and/or interleukin receptor encoding sequence. In one embodiment, the invention features an siNA molecule that down-regulates expression of a interleukin and/or interleukin receptor gene, for example, wherein the interleukin and/or interleukin receptor gene comprises interleukin and/or interleukin receptor non-coding sequence or regulatory elements involved in interleukin and/or interleukin receptor gene expression.

In one embodiment, an siNA of the invention is used to inhibit the expression of interleukin and/or interleukin receptor genes or a interleukin and/or interleukin receptor gene family, wherein the genes or gene family sequences share sequence homology. Such homologous sequences can be identified as is known in the art, for example using sequence alignments. siNA molecules can be designed to target such homologous sequences, for example using perfectly complementary sequences or by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, that can provide additional target sequences. In instances where mismatches are identified, non-canonical base pairs, for example mismatches and/or wobble bases, can be used to generate siNA molecules that target both more than one gene sequences. In a non-limiting example, non-canonical base pairs such as UU and CC base pairs are used to generate siNA molecules that are capable of targeting sequences for differing interleukin and/or interleukin receptor targets that share sequence homology (e.g., differing interleukin genes or differing allelic variants thereof). As such, one advantage of using siNAs of the invention is that a single siNA can be designed to include nucleic acid sequence that is complementary to the nucleotide sequence that is conserved between the homologous genes. In this approach, a single siNA can be used to inhibit expression of more than one interleukin and/or interleukin receptor gene instead of using more than one siNA molecule to target the different genes.

In one embodiment, the invention features an siNA molecule having RNAi activity against interleukin and/or interleukin receptor RNA, wherein the siNA molecule comprises a sequence complementary to any RNA having interleukin and/or interleukin receptor encoding sequence, such as those sequences having GenBank Accession Nos. shown in Table I. In another embodiment, the invention features an siNA molecule having RNAi activity against interleukin and/or interleukin receptor RNA, wherein the siNA molecule comprises a sequence complementary to an RNA having variant interleukin and/or interleukin receptor encoding sequence, for example other mutant interleukin and/or interleukin receptor genes not shown in Table I but known in the art to be associated with diseases, traits, or conditions described herein or otherwise known in the art. Chemical modifications as shown in Tables III and IV or otherwise described herein can be applied to any siNA construct of the invention. In another embodiment, an siNA molecule of the invention includes a nucleotide sequence that can interact with nucleotide sequence of a interleukin and/or interleukin receptor gene and thereby mediate silencing of interleukin and/or interleukin receptor gene expression, for example, wherein the siNA mediates regulation of interleukin and/or interleukin receptor gene expression by cellular processes that modulate the chromatin structure or methylation patterns of the interleukin and/or interleukin receptor gene and prevent transcription of the interleukin and/or interleukin receptor gene.

In one embodiment, siNA molecules of the invention are used to down regulate or inhibit the expression of interleukin and/or interleukin receptor proteins arising from interleukin and/or interleukin receptor haplotype polymorphisms that are associated with a disease or condition, (e.g., proliferative, inflammatory, autoimmune, respiratory, pulmonary, cardiovascular, neurodegenerative diseases). Analysis of interleukin and/or interleukin receptor genes, or interleukin and/or interleukin receptor protein or RNA levels can be used to identify subjects with such polymorphisms or those subjects who are at risk of developing traits, conditions, or diseases described herein. These subjects are amenable to treatment, for example, treatment with siNA molecules of the invention and any other composition useful in treating diseases related to interleukin and/or interleukin receptor gene expression. As such, analysis of interleukin and/or interleukin receptor protein or RNA levels can be used to determine treatment type and the course of therapy in treating a subject. Monitoring of interleukin and/or interleukin receptor protein or RNA levels can be used to predict treatment outcome and to determine the efficacy of compounds and compositions that modulate the level and/or activity of certain interleukin and/or interleukin receptor proteins associated with a trait, condition, or disease.

In one embodiment of the invention an siNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a interleukin and/or interleukin receptor protein. The siNA further comprises a sense strand, wherein said sense strand comprises a nucleotide sequence of a interleukin and/or interleukin receptor gene or a portion thereof.

In another embodiment, the invention features an siNA molecule comprising a nucleotide sequence in the antisense region of the siNA molecule that is complementary to a nucleotide sequence or portion of sequence of a interleukin and/or interleukin receptor gene. In another embodiment, the invention features an siNA molecule comprising a region, for example, the antisense region of the siNA construct, complementary to a sequence comprising a interleukin and/or interleukin receptor gene sequence or a portion thereof.

In one embodiment, the antisense region of interleukin and/or interleukin receptor siNA constructs comprises a sequence complementary to sequence having any of SEQ ID NOs. 1-81, 163-213, 265-464, 665-735, 807-1029, or 1253-1260. In one embodiment, the antisense region of interleukin and/or interleukin receptor constructs comprises sequence having any of SEQ ID NOs. 82-162, 214-264, 465-664, 736-806, 1030-1252, 1319-1326, 1335-1342, 1351-1358, 1367-1374, 1383-1406, 1415-1422, 1431-1438, 1447-1454, 1463-1470, 1479-1502, 1511-1518, 1527-1534, 1543-1550, 1559-1566, 1575-1598, 1607-1614, 1623-1630, 1649-1656, 1665-1682, 1691-1714, 1723-1730, 1739-1746, 1755-1762, 1771-1778, 1787-1810, 1812, 1814, 1816, 1819, 1821, 1823, 1825, or 1828. In another embodiment, the sense region of interleukin and/or interleukin receptor constructs comprises sequence having any of SEQ ID NOs. 1-81, 163-213, 265-464, 665-735, 807-1029, 1253-1260, 1311-1318, 1327-1334, 1343-1350, 1359-1366, 1375-1382, 1269-1276, 1407-1414, 1423-1430, 1439-1446, 1455-1462, 1471-1478, 1277-1284, 1503-1510, 1519-1526, 1535-1542, 1551-1558, 1567-1574, 1285-1292, 1599-1606, 1615-1622, 1631-1648, 1657-1664, 1683-1690, 1303-1310, 1715-1722, 1731-1738, 1747-1754, 1763-1770, 1779-1786, 1811, 1813, 1815, 1817, 1818, 1820, 1822, 1824, 1826, or 1827.

In one embodiment, an siNA molecule of the invention comprises any of SEQ ID NOs. 1-1828. The sequences shown in SEQ ID NOs: 1-1828 are not limiting. An siNA molecule of the invention can comprise any contiguous interleukin and/or interleukin receptor sequence (e.g., about 19 to about 25, or about 19, 20, 21, 22, 23, 24, or 25 contiguous interleukin and/or interleukin receptor nucleotides).

In yet another embodiment, the invention features an siNA molecule comprising a sequence, for example, the antisense sequence of the siNA construct, complementary to a sequence or portion of sequence comprising sequence represented by GenBank Accession Nos. shown in Table I. Chemical modifications in Tables III and IV and described herein can be applied to any siNA construct of the invention.

In one embodiment of the invention an siNA molecule comprises an antisense strand having about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides, wherein the antisense strand is complementary to a RNA sequence encoding a interleukin and/or interleukin receptor protein, and wherein said siNA further comprises a sense strand having about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides, and wherein said sense strand and said antisense strand are distinct nucleotide sequences with at least about 19 complementary nucleotides.

In another embodiment of the invention an siNA molecule of the invention comprises an antisense region having about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides, wherein the antisense region is complementary to a RNA sequence encoding a interleukin and/or interleukin receptor protein, and wherein said siNA further comprises a sense region having about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides, wherein said sense region and said antisense region comprise a linear molecule with at least about 19 complementary nucleotides.

In one embodiment, an siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a interleukin and/or interleukin receptor gene. Because interleukin and/or interleukin receptor genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of interleukin and/or interleukin receptor genes or alternately specific interleukin and/or interleukin receptor genes (e.g., polymorphic variants) by selecting sequences that are either shared amongst different interleukin and/or interleukin receptor targets or alternatively that are unique for a specific interleukin and/or interleukin receptor target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of interleukin and/or interleukin receptor RNA sequences having homology among several interleukin and/or interleukin receptor gene variants so as to target a class of interleukin and/or interleukin receptor genes with one siNA molecule. Accordingly, in one embodiment, the siNA molecule of the invention modulates the expression of one or both interleukin and/or interleukin receptor alleles in a subject. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific interleukin and/or interleukin receptor RNA sequence (e.g., a single interleukin and/or interleukin receptor allele or interleukin and/or interleukin receptor single nucleotide polymorphism (SNP)) due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.

In one embodiment, nucleic acid molecules of the invention that act as mediators of the RNA interference gene silencing response are double-stranded nucleic acid molecules. In another embodiment, the siNA molecules of the invention consist of duplex nucleic acid molecules containing about 19 base pairs between oligonucleotides comprising about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides. In yet another embodiment, siNA molecules of the invention comprise duplex nucleic acid molecules with overhanging ends of about 1 to about 3 (e.g., about 1, 2, or 3) nucleotides, for example, about 21-nucleotide duplexes with about 19 base pairs and 3′-terminal mononucleotide, dinucleotide, or trinucleotide overhangs.

In one embodiment, the invention features one or more chemically-modified siNA constructs having specificity for interleukin and/or interleukin receptor expressing nucleic acid molecules, such as RNA encoding a interleukin and/or interleukin receptor protein. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation. These chemical modifications, when used in various siNA constructs, are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al., supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well-tolerated and confer substantial increases in serum stability for modified siNA constructs.

In one embodiment, an siNA molecule of the invention comprises modified nucleotides while maintaining the ability to mediate RNAi. The modified nucleotides can be used to improve in vitro or in vivo characteristics such as stability, activity, and/or bioavailability. For example, an siNA molecule of the invention can comprise modified nucleotides as a percentage of the total number of nucleotides present in the siNA molecule. As such, an siNA molecule of the invention can generally comprise about 5% to about 100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentage of modified nucleotides present in a given siNA molecule will depend on the total number of nucleotides present in the siNA. If the siNA molecule is single-stranded, the percent modification can be based upon the total number of nucleotides present in the single-stranded siNA molecules. Likewise, if the siNA molecule is double-stranded, the percent modification can be based upon the total number of nucleotides present in the sense strand, antisense strand, or both the sense and antisense strands.

One aspect of the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a interleukin and/or interleukin receptor gene. In one embodiment, the double-stranded siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is about 21 nucleotides long. In one embodiment, the double-stranded siNA molecule does not contain any ribonucleotides. In another embodiment, the double-stranded siNA molecule comprises one or more ribonucleotides. In one embodiment, each strand of the double-stranded siNA molecule comprises about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides, wherein each strand comprises about 19 nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof of the interleukin and/or interleukin receptor gene, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the interleukin and/or interleukin receptor gene or a portion thereof.

In another embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a interleukin and/or interleukin receptor gene comprising an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of the interleukin and/or interleukin receptor gene or a portion thereof, and a sense region, wherein the sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of the interleukin and/or interleukin receptor gene or a portion thereof. In one embodiment, the antisense region and the sense region each comprise about 19 to about 23 (e.g. about 19, 20, 21, 22, or 23) nucleotides, wherein the antisense region comprises about 19 nucleotides that are complementary to nucleotides of the sense region.

In another embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a interleukin and/or interleukin receptor gene comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the interleukin and/or interleukin receptor gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region.

In one embodiment, an siNA molecule of the invention comprises blunt ends, i.e., ends that do not include any overhanging nucleotides. For example, an siNA molecule comprising modifications described herein (e.g., comprising nucleotides having Formulae I-VII or siNA constructs comprising “Stab 00”-“Stab 25” (Table IV) or any combination thereof (see Table IV)) and/or any length described herein can comprise blunt ends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise one or more blunt ends, i.e. where a blunt end does not have any overhanging nucleotides. In one embodiment, the blunt ended siNA molecule has a number of base pairs equal to the number of nucleotides present in each strand of the siNA molecule. In another embodiment, the siNA molecule comprises one blunt end, for example wherein the 5′-end of the antisense strand and the 3′-end of the sense strand do not have any overhanging nucleotides. In another example, the siNA molecule comprises one blunt end, for example wherein the 3′-end of the antisense strand and the 5′-end of the sense strand do not have any overhanging nucleotides. In another example, an siNA molecule comprises two blunt ends, for example wherein the 3′-end of the antisense strand and the 5′-end of the sense strand as well as the 5′-end of the antisense strand and 3′-end of the sense strand do not have any overhanging nucleotides. A blunt ended siNA molecule can comprise, for example, from about 18 to about 30 nucleotides (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). Other nucleotides present in a blunt ended siNA molecule can comprise mismatches, bulges, loops, or wobble base pairs, for example, to modulate the activity of the siNA molecule to mediate RNA interference.

By “blunt ends” is meant symmetric termini or termini of a double-stranded siNA molecule having no overhanging nucleotides. The two strands of a double-stranded siNA molecule align with each other without over-hanging nucleotides at the termini. For example, a blunt ended siNA construct comprises terminal nucleotides that are complementary between the sense and antisense regions of the siNA molecule.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a interleukin and/or interleukin receptor gene, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. The sense region can be connected to the antisense region via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker.

In one embodiment, the invention features double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a interleukin and/or interleukin receptor gene, wherein the siNA molecule comprises about 19 to about 21 base pairs, and wherein each strand of the siNA molecule comprises one or more chemical modifications. In another embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a interleukin and/or interleukin receptor gene or a portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of the interleukin and/or interleukin receptor gene. In another embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a interleukin and/or interleukin receptor gene or portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or portion thereof of the interleukin and/or interleukin receptor gene. In another embodiment, each strand of the siNA molecule comprises about 19 to about 23 nucleotides, and each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand. The interleukin and/or interleukin receptor gene can comprise, for example, sequences referred to in Table I.

In one embodiment, an siNA molecule of the invention comprises no ribonucleotides. In another embodiment, an siNA molecule of the invention comprises ribonucleotides.

In one embodiment, an siNA molecule of the invention comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of a interleukin and/or interleukin receptor gene or a portion thereof, and the siNA further comprises a sense region comprising a nucleotide sequence substantially similar to the nucleotide sequence of the interleukin and/or interleukin receptor gene or a portion thereof. In another embodiment, the antisense region and the sense region each comprise about 19 to about 23 nucleotides and the antisense region comprises at least about 19 nucleotides that are complementary to nucleotides of the sense region. The interleukin and/or interleukin receptor gene can comprise, for example, sequences referred to in Table I.

In one embodiment, an siNA molecule of the invention comprises a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by a interleukin and/or interleukin receptor gene, or a portion thereof, and the sense region comprises a nucleotide sequence that is complementary to the antisense region. In one embodiment, the siNA molecule is assembled from two separate oligonucleotide fragments, wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. In another embodiment, the sense region is connected to the antisense region via a linker molecule. In another embodiment, the sense region is connected to the antisense region via a linker molecule, such as a nucleotide or non-nucleotide linker. The interleukin and/or interleukin receptor gene can comprise, for example, sequences referred in to Table I.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a interleukin and/or interleukin receptor gene comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the interleukin and/or interleukin receptor gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the siNA molecule has one or more modified pyrimidine and/or purine nucleotides. In one embodiment, the pyrimidine nucleotides in the sense region are 2′-O-methylpyrimidine nucleotides or 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides. In another embodiment, the pyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In one embodiment, the pyrimidine nucleotides in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the antisense region are 2′-O-methyl or 2′-deoxy purine nucleotides. In another embodiment of any of the above-described siNA molecules, any nucleotides present in a non-complementary region of the sense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a interleukin and/or interleukin receptor gene, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule, and wherein the fragment comprising the sense region includes a terminal cap moiety at the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the fragment. In one embodiment, the terminal cap moiety is an inverted deoxy abasic moiety or glyceryl moiety. In one embodiment, each of the two fragments of the siNA molecule comprise about 21 nucleotides.

In one embodiment, the invention features an siNA molecule comprising at least one modified nucleotide, wherein the modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide. The siNA can be, for example, of length between about 12 and about 36 nucleotides. In one embodiment, all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment, all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. The siNA can further comprise at least one modified internucleotidic linkage, such as phosphorothioate linkage. In one embodiment, the 2′-deoxy-2′-fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a method of increasing the stability of an siNA molecule against cleavage by ribonucleases comprising introducing at least one modified nucleotide into the siNA molecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide. In one embodiment, all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment, all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. The siNA can further comprise at least one modified internucleotidic linkage, such as phosphorothioate linkage. In one embodiment, the 2′-deoxy-2′-fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a interleukin and/or interleukin receptor gene comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the interleukin and/or interleukin receptor gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the purine nucleotides present in the antisense region comprise 2′-deoxy-purine nucleotides. In an alternative embodiment, the purine nucleotides present in the antisense region comprise 2′-O-methyl purine nucleotides. In either of the above embodiments, the antisense region can comprise a phosphorothioate internucleotide linkage at the 3′ end of the antisense region. Alternatively, in either of the above embodiments, the antisense region can comprise a glyceryl modification at the 3′ end of the antisense region. In another embodiment of any of the above-described siNA molecules, any nucleotides present in a non-complementary region of the antisense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the antisense region of an siNA molecule of the invention comprises sequence complementary to a portion of a interleukin and/or interleukin receptor transcript having sequence unique to a particular interleukin and/or interleukin receptor disease related allele, such as sequence comprising a single nucleotide polymorphism (SNP) associated with the disease specific allele. As such, the antisense region of an siNA molecule of the invention can comprise sequence complementary to sequences that are unique to a particular allele to provide specificity in mediating selective RNAi against the disease, condition, or trait related allele.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a interleukin and/or interleukin receptor gene, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. In another embodiment about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule and wherein at least two 3′ terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, such as a 2′-deoxy-thymidine. In another embodiment, all 21 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule. In another embodiment, about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the interleukin and/or interleukin receptor gene. In another embodiment, about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the interleukin and/or interleukin receptor gene. In any of the above embodiments, the 5′-end of the fragment comprising said antisense region can optionally include a phosphate group.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a interleukin and/or interleukin receptor RNA sequence (e.g., wherein said target RNA sequence is encoded by a interleukin and/or interleukin receptor gene involved in the interleukin and/or interleukin receptor pathway), wherein the siNA molecule does not contain any ribonucleotides and wherein each strand of the double-stranded siNA molecule is about 21 nucleotides long. Examples of non-ribonucleotide containing siNA constructs are combinations of stabilization chemistries shown in Table IV in any combination of Sense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, or Stab 18/20.

In one embodiment, the invention features a chemically synthesized double-stranded RNA molecule that directs cleavage of a interleukin and/or interleukin receptor RNA via RNA interference, wherein each strand of said RNA molecule is about 21 to about 23 nucleotides in length; one strand of the RNA molecule comprises nucleotide sequence having sufficient complementarity to the interleukin and/or interleukin receptor RNA for the RNA molecule to direct cleavage of the interleukin and/or interleukin receptor RNA via RNA interference; and wherein at least one strand of the RNA molecule comprises one or more chemically modified nucleotides described herein, such as deoxynucleotides, 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-O-methoxyethyl nucleotides etc.

In one embodiment, the invention features a medicament comprising an siNA molecule of the invention.

In one embodiment, the invention features an active ingredient comprising an siNA molecule of the invention.

In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule to down-regulate expression of a interleukin and/or interleukin receptor gene, wherein the siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is about 18 to about 28 or more (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 or more) nucleotides long.

In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a interleukin and/or interleukin receptor gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of interleukin and/or interleukin receptor RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a interleukin and/or interleukin receptor gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of interleukin and/or interleukin receptor RNA or a portion thereof, wherein the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a interleukin and/or interleukin receptor gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of interleukin and/or interleukin receptor RNA that encodes a protein or portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, each strand of the siNA molecule comprises about 18 to about 29 or more (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 or more) nucleotides, wherein each strand comprises at least about 18 nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, the siNA molecule is assembled from two oligonucleotide fragments, wherein one fragment comprises the nucleotide sequence of the antisense strand of the siNA molecule and a second fragment comprises nucleotide sequence of the sense region of the siNA molecule. In one embodiment, the sense strand is connected to the antisense strand via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker. In a further embodiment, the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides. In still another embodiment, the pyrimidine nucleotides present in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2′-deoxy purine nucleotides. In another embodiment, the antisense strand comprises one or more 2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methyl purine nucleotides. In another embodiment, the pyrimidine nucleotides present in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2′-O-methyl purine nucleotides. In a further embodiment the sense strand comprises a 3′-end and a 5′-end, wherein a terminal cap moiety (e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotide moiety such as inverted thymidine) is present at the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the sense strand. In another embodiment, the antisense strand comprises a phosphorothioate internucleotide linkage at the 3′ end of the antisense strand. In another embodiment, the antisense strand comprises a glyceryl modification at the 3′ end. In another embodiment, the 5′-end of the antisense strand optionally includes a phosphate group.

In any of the above-described embodiments of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a interleukin and/or interleukin receptor gene, wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, each of the two strands of the siNA molecule can comprise about 21 nucleotides. In one embodiment, about 21 nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule. In another embodiment, about 19 nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule, wherein at least two 3′ terminal nucleotides of each strand of the siNA molecule are not base-paired to the nucleotides of the other strand of the siNA molecule. In another embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine. In one embodiment, each strand of the siNA molecule is base-paired to the complementary nucleotides of the other strand of the siNA molecule. In one embodiment, about 19 nucleotides of the antisense strand are base-paired to the nucleotide sequence of the interleukin and/or interleukin receptor RNA or a portion thereof. In one embodiment, about 21 nucleotides of the antisense strand are base-paired to the nucleotide sequence of the interleukin and/or interleukin receptor RNA or a portion thereof.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a interleukin and/or interleukin receptor gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of interleukin and/or interleukin receptor RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the 5′-end of the antisense strand optionally includes a phosphate group.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a interleukin and/or interleukin receptor gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of interleukin and/or interleukin receptor RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the nucleotide sequence or a portion thereof of the antisense strand is complementary to a nucleotide sequence of the untranslated region or a portion thereof of the interleukin and/or interleukin receptor RNA.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a interleukin and/or interleukin receptor gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of interleukin and/or interleukin receptor RNA or a portion thereof, wherein the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand, wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the nucleotide sequence of the antisense strand is complementary to a nucleotide sequence of the interleukin and/or interleukin receptor RNA or a portion thereof that is present in the interleukin and/or interleukin receptor RNA.

In one embodiment, the invention features a composition comprising an siNA molecule of the invention in a pharmaceutically acceptable carrier or diluent.

In a non-limiting example, the introduction of chemically-modified nucleotides into nucleic acid molecules provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously. For example, the use of chemically-modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically-modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically-modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example, when compared to an all-RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule can be greater than that of the native molecule due to improved stability and/or delivery of the molecule. Unlike native unmodified siNA, chemically-modified siNA can also minimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, the antisense region of an siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the antisense region can comprise about one to about five phosphorothioate internucleotide linkages at the 5′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs of an siNA molecule of the invention can comprise ribonucleotides or deoxyribonucleotides that are chemically-modified at a nucleic acid sugar, base, or backbone. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more universal base ribonucleotides. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.

One embodiment of the invention provides an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention in a manner that allows expression of the nucleic acid molecule. Another embodiment of the invention provides a mammalian cell comprising such an expression vector. The mammalian cell can be a human cell. The siNA molecule of the expression vector can comprise a sense region and an antisense region. The antisense region can comprise sequence complementary to a RNA or DNA sequence encoding interleukin and/or interleukin receptor and the sense region can comprise sequence complementary to the antisense region. The siNA molecule can comprise two distinct strands having complementary sense and antisense regions. The siNA molecule can comprise a single strand having complementary sense and antisense regions.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against interleukin and/or interleukin receptor inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising a backbone modified internucleotide linkage having Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally-occurring or chemically-modified, each X and Y is independently O, S, N, alkyl, or substituted alkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl and wherein W, X, Y, and Z are optionally not all O. In another embodiment, a backbone modification of the invention comprises a phosphonoacetate and/or thiophosphonoacetate internucleotide linkage (see for example Sheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically-modified internucleotide linkages having Formula I, for example, wherein any Z, W, X, and/or Y independently comprises a sulphur atom, can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modified internucleotide linkages having Formula I at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified internucleotide linkages having Formula I at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In another embodiment, an siNA molecule of the invention having internucleotide linkage(s) of Formula I also comprises a chemically-modified nucleotide or non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against interleukin and/or interleukin receptor inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula II can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotide or non-nucleotide of Formula II at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 5′-end of the sense strand, the antisense strand, or both strands. In anther non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 3′-end of the sense strand, the antisense strand, or both strands.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against interleukin and/or interleukin receptor inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be employed to be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula III can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotide or non-nucleotide of Formula III at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) or non-nucleotide(s) of Formula III at the 5′-end of the sense strand, the antisense strand, or both strands. In anther non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide of Formula III at the 3′-end of the sense strand, the antisense strand, or both strands.

In another embodiment, an siNA molecule of the invention comprises a nucleotide having Formula II or III, wherein the nucleotide having Formula II or III is in an inverted configuration. For example, the nucleotide having Formula II or III is connected to the siNA construct in a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against interleukin and/or interleukin receptor inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a 5′-terminal phosphate group having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl, or alkylhalo; wherein each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, or acetyl; and wherein W, X, Y and Z are not all O.

In one embodiment, the invention features an siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand, for example, a strand complementary to a target RNA, wherein the siNA molecule comprises an all RNA siNA molecule. In another embodiment, the invention features an siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand wherein the siNA molecule also comprises about 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminal nucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or 4) deoxyribonucleotides on the 3′-end of one or both strands. In another embodiment, a 5′-terminal phosphate group having Formula IV is present on the target-complementary strand of an siNA molecule of the invention, for example an siNA molecule having chemical modifications having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against interleukin and/or interleukin receptor inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more phosphorothioate internucleotide linkages. For example, in a non-limiting example, the invention features a chemically-modified short interfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one siNA strand. In yet another embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both siNA strands. The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more phosphorothioate internucleotide linkages at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate internucleotide linkages at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands.

In one embodiment, the invention features an siNA molecule, wherein the sense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, wherein the sense strand comprises about 1 to about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5 or more, for example about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features an siNA molecule, wherein the antisense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule having about 1 to about 5, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features an siNA molecule comprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one or both siNA sequence strands. In addition, the 2′-5′ internucleotide linkage(s) can be present at various other positions within one or both siNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a pyrimidine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a purine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified, wherein each strand is about 18 to about 27 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides in length, wherein the duplex has about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the chemical modification comprises a structure having any of Formulae I-VII. For example, an exemplary chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein each strand consists of about 21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotide overhang, and wherein the duplex has about 19 base pairs. In another embodiment, an siNA molecule of the invention comprises a single-stranded hairpin structure, wherein the siNA is about 36 to about 70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siNA can include a chemical modification comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 19 base pairs and a 2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. For example, a linear hairpin siNA molecule of the invention is designed such that degradation of the loop portion of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, an siNA molecule of the invention comprises a hairpin structure, wherein the siNA is about 25 to about 50 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 3 to about 23 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23) base pairs and a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV). In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. In one embodiment, a linear hairpin siNA molecule of the invention comprises a loop portion comprising a non-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises an asymmetric hairpin structure, wherein the siNA is about 25 to about 50 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3 to about 20 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) base pairs, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms an asymmetric hairpin structure having about 3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) base pairs and a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV). In one embodiment, an asymmetric hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. In another embodiment, an asymmetric hairpin siNA molecule of the invention comprises a loop portion comprising a non-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises an asymmetric double-stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 16 to about 25 (e.g., about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in length, wherein the sense region is about 3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotides in length, wherein the sense region and the antisense region have at least 3 complementary nucleotides, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises an asymmetric double-stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 18 to about 22 (e.g., about 18, 19, 20, 21, or 22) nucleotides in length and wherein the sense region is about 3 to about 15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) nucleotides in length, wherein the sense region the antisense region have at least 3 complementary nucleotides, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. In another embodiment, the asymmetric double-stranded siNA molecule can also have a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV).

In another embodiment, an siNA molecule of the invention comprises a circular nucleic acid molecule, wherein the siNA is about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siNA can include a chemical modification, which comprises a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a circular oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the invention contains two loop motifs, wherein one or both loop portions of the siNA molecule is biodegradable. For example, a circular siNA molecule of the invention is designed such that degradation of the loop portions of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In one embodiment, an siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety, for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, or group having Formula I or II; and R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, an siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasic moiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and either R5, R3, R8 or R13 serves as a point of attachment to the siNA molecule of the invention.

In another embodiment, an siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substituted polyalkyl moieties, for example a compound having Formula VII:

wherein each n is independently an integer from 1 to 12, each R1, R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, or a group having Formula I, and R1, R2 or R3 serves as points of attachment to the siNA molecule of the invention.

In another embodiment, the invention features a compound having Formula VII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises O and is the point of attachment to the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both strands of a double-stranded siNA molecule of the invention or to a single-stranded siNA molecule of the invention. This modification is referred to herein as “glyceryl” (for example modification 6 in FIG. 10).

In another embodiment, a moiety having any of Formula V, VI or VII of the invention is at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of an siNA molecule of the invention. For example, a moiety having Formula V, VI or VII can be present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense strand, the sense strand, or both antisense and sense strands of the siNA molecule. In addition, a moiety having Formula VII can be present at the 3′-end or the 5′-end of a hairpin siNA molecule as described herein.

In another embodiment, an siNA molecule of the invention comprises an abasic residue having Formula V or VI, wherein the abasic residue having Formula VI or VI is connected to the siNA construct in a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, an siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (LNA) nucleotides, for example at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, an siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides, for example at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against interleukin and/or interleukin receptor inside a cell or reconstituted in vitro system comprising a sense region, wherein one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), and an antisense region, wherein one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides). The sense region and/or the antisense region can have a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense and/or antisense sequence. The sense and/or antisense region can optionally further comprise a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhang nucleotides can further comprise one or more (e.g., about 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown in FIGS. 4 and 5 and Tables III and IV herein. In any of these described embodiments, the purine nucleotides present in the sense region are alternatively 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides) and one or more purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides). Also, in any of these embodiments, one or more purine nucleotides present in the sense region are alternatively purine ribonucleotides (e.g., wherein all purine nucleotides are purine ribonucleotides or alternately a plurality of purine nucleotides are purine ribonucleotides) and any purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides). Additionally, in any of these embodiments, one or more purine nucleotides present in the sense region and/or present in the antisense region are alternatively selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g., wherein all purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides or alternately a plurality of purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, are resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi. Non-limiting examples of nucleotides having a Northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double-stranded siNA molecule of the invention comprises a terminal cap moiety, (see for example FIG. 10) such as an inverted deoxyabasic moiety, at the 3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi) against interleukin and/or interleukin receptor inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a conjugate covalently attached to the chemically-modified siNA molecule. Non-limiting examples of conjugates contemplated by the invention include conjugates and ligands described in Vargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003, incorporated by reference herein in its entirety, including the drawings. In another embodiment, the conjugate is covalently attached to the chemically-modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In another embodiment, the conjugate molecule is attached at the 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In yet another embodiment, the conjugate molecule is attached both the 3′-end and 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule, or any combination thereof. In one embodiment, a conjugate molecule of the invention comprises a molecule that facilitates delivery of a chemically-modified siNA molecule into a biological system, such as a cell. In another embodiment, the conjugate molecule attached to the chemically-modified siNA molecule is a polyethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to chemically-modified siNA molecules are described in Vargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002 incorporated by reference herein. The type of conjugates used and the extent of conjugation of siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of siNA constructs while at the same time maintaining the ability of the siNA to mediate RNAi activity. As such, one skilled in the art can screen siNA constructs that are modified with various conjugates to determine whether the siNA conjugate complex possesses improved properties while maintaining the ability to mediate RNAi, for example in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule of the invention, wherein the siNA further comprises a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the siNA to the antisense region of the siNA. In one embodiment, a nucleotide linker of the invention can be a linker of ≧2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In another embodiment, the nucleotide linker can be a nucleic acid aptamer. By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has a sequence that comprises a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art. (See, for example, Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the invention comprises abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemisty 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No. WO 89/02439; Usman et al., International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein. A “non-nucleotide” further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, for example at the C1 position of the sugar.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein one or both strands of the siNA molecule that are assembled from two separate oligonucleotides do not comprise any ribonucleotides. For example, an siNA molecule can be assembled from a single oligonucleotide where the sense and antisense regions of the siNA comprise separate oligonucleotides that do not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotides. In another example, an siNA molecule can be assembled from a single oligonucleotide where the sense and antisense regions of the siNA are linked or circularized by a nucleotide or non-nucleotide linker as described herein, wherein the oligonucleotide does not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotide. Applicant has surprisingly found that the presence of ribonucleotides (e.g., nucleotides having a 2′-hydroxyl group) within the siNA molecule is not required or essential to support RNAi activity. As such, in one embodiment, all positions within the siNA can include chemically modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having Formula I, II, III, IV, V, VI, or VII or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

In one embodiment, an siNA molecule of the invention is a single-stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system comprising a single-stranded polynucleotide having complementarity to a target nucleic acid sequence. In another embodiment, the single-stranded siNA molecule of the invention comprises a 5′-terminal phosphate group. In another embodiment, the single-stranded siNA molecule of the invention comprises a 5′-terminal phosphate group and a 3′-terminal phosphate group (e.g., a 2′,3′-cyclic phosphate). In another embodiment, the single-stranded siNA molecule of the invention comprises about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides. In yet another embodiment, the single-stranded siNA molecule of the invention comprises one or more chemically modified nucleotides or non-nucleotides described herein. For example, all the positions within the siNA molecule can include chemically-modified nucleotides such as nucleotides having any of Formulae I-VII, or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

In one embodiment, an siNA molecule of the invention is a single-stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system comprising a single-stranded polynucleotide having complementarity to a target nucleic acid sequence, wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end and/or the 5′-end. The siNA optionally further comprises about 1 to about 4 or more (e.g., about 1, 2, 3, 4 or more) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group. In any of these embodiments, any purine nucleotides present in the antisense region are alternatively 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides). Also, in any of these embodiments, any purine nucleotides present in the siNA (i.e., purine nucleotides present in the sense and/or antisense region) can alternatively be locked nucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides or alternately a plurality of purine nucleotides are LNA nucleotides). Also, in any of these embodiments, any purine nucleotides present in the siNA are alternatively 2′-methoxyethyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-methoxyethyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-methoxyethyl purine nucleotides). In another embodiment, any modified nucleotides present in the single-stranded siNA molecules of the invention comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the single-stranded siNA molecules of the invention are preferably resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi.

In one embodiment, the invention features a method for modulating the expression of a interleukin and/or interleukin receptor gene within a cell comprising: (a) synthesizing an siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the interleukin and/or interleukin receptor gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the cell.

In one embodiment, the invention features a method for modulating the expression of a interleukin and/or interleukin receptor gene within a cell comprising: (a) synthesizing an siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the interleukin and/or interleukin receptor gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one interleukin and/or interleukin receptor gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the interleukin and/or interleukin receptor genes; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor genes in the cell.

In another embodiment, the invention features a method for modulating the expression of two or more interleukin and/or interleukin receptor genes within a cell comprising: (a) synthesizing one or more siNA molecules of the invention, which can be chemically-modified, wherein the siNA strands comprise sequences complementary to RNA of the interleukin and/or interleukin receptor genes and wherein the sense strand sequences of the siNAs comprise sequences identical or substantially similar to the sequences of the target RNAs; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor genes in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one interleukin and/or interleukin receptor gene within a cell comprising: (a) synthesizing an siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the interleukin and/or interleukin receptor gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the target RNAs; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor genes in the cell.

In one embodiment, siNA molecules of the invention are used as reagents in ex vivo applications. For example, siNA reagents are introduced into tissue or cells that are transplanted into a subject for therapeutic effect. The cells and/or tissue can be derived from an organism or subject that later receives the explant, or can be derived from another organism or subject prior to transplantation. The siNA molecules can be used to modulate the expression of one or more genes in the cells or tissue, such that the cells or tissue obtain a desired phenotype or are able to perform a function when transplanted in vivo. In one embodiment, certain target cells from a patient are extracted. These extracted cells are contacted with siNAs targeting a specific nucleotide sequence within the cells under conditions suitable for uptake of the siNAs by these cells (e.g. using delivery reagents such as cationic lipids, liposomes and the like or using techniques such as electroporation to facilitate the delivery of siNAs into cells). The cells are then reintroduced back into the same patient or other patients. In one embodiment, the invention features a method of modulating the expression of a interleukin and/or interleukin receptor gene in a tissue explant comprising: (a) synthesizing an siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the interleukin and/or interleukin receptor gene; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in that organism.

In one embodiment, the invention features a method of modulating the expression of a interleukin and/or interleukin receptor gene in a tissue explant comprising: (a) synthesizing an siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the interleukin and/or interleukin receptor gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in that organism.

In another embodiment, the invention features a method of modulating the expression of more than one interleukin and/or interleukin receptor gene in a tissue explant comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the interleukin and/or interleukin receptor genes; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor genes in that organism.

In one embodiment, the invention features a method of modulating the expression of a interleukin and/or interleukin receptor gene in an organism comprising: (a) synthesizing an siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the interleukin and/or interleukin receptor gene; and (b) introducing the siNA molecule into the organism under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism. The level of interleukin and/or interleukin receptor protein or RNA can be determined as is known in the art.

In another embodiment, the invention features a method of modulating the expression of more than one interleukin and/or interleukin receptor gene in an organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the interleukin and/or interleukin receptor genes; and (b) introducing the siNA molecules into the organism under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor genes in the organism. The level of interleukin and/or interleukin receptor protein or RNA can be determined as is known in the art.

In one embodiment, the invention features a method for modulating the expression of a interleukin and/or interleukin receptor gene within a cell comprising: (a) synthesizing an siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single-stranded sequence having complementarity to RNA of the interleukin and/or interleukin receptor gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one interleukin and/or interleukin receptor gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single-stranded sequence having complementarity to RNA of the interleukin and/or interleukin receptor gene; and (b) contacting the cell in vitro or in vivo with the siNA molecule under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor genes in the cell.

In one embodiment, the invention features a method of modulating the expression of a interleukin and/or interleukin receptor gene in a tissue explant comprising: (a) synthesizing an siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single-stranded sequence having complementarity to RNA of the interleukin and/or interleukin receptor gene; and (b) contacting the cell of the tissue explant derived from a particular organism with the siNA molecule under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in that organism.

In another embodiment, the invention features a method of modulating the expression of more than one interleukin and/or interleukin receptor gene in a tissue explant comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single-stranded sequence having complementarity to RNA of the interleukin and/or interleukin receptor gene; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor genes in that organism.

In one embodiment, the invention features a method of modulating the expression of a interleukin and/or interleukin receptor gene in an organism comprising: (a) synthesizing an siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single-stranded sequence having complementarity to RNA of the interleukin and/or interleukin receptor gene; and (b) introducing the siNA molecule into the organism under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism.

In another embodiment, the invention features a method of modulating the expression of more than one interleukin and/or interleukin receptor gene in an organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single-stranded sequence having complementarity to RNA of the interleukin and/or interleukin receptor gene; and (b) introducing the siNA molecules into the organism under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor genes in the organism.

In one embodiment, the invention features a method of modulating the expression of a interleukin and/or interleukin receptor gene in an organism comprising contacting the organism with an siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating or preventing a disease, condition, trait, genotype or phenotype in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease, condition, trait, genotype or phenotype in the subject, alone or in conjunction with one or more other therapeutic compounds. In yet another embodiment, the invention features a method for reducing or preventing tissue rejection in a subject comprising administering to the subject a composition of the invention under conditions suitable for the reduction or prevention of tissue rejection in the subject.

In one embodiment, the invention features a method for treating an inflammatory disease or condition in an organism comprising contacting the organism with an siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating or preventing an allergic reaction, disease, or condition in an organism comprising contacting the organism with an siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating or preventing an autoimmune disease or condition in an organism comprising contacting the organism with an siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating or preventing cancer in an organism comprising contacting the organism with an siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating or preventing a respiratory disease or condition in an organism comprising contacting the organism with an siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating or preventing a pulmonary disease or condition in an organism comprising contacting the organism with an siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating or preventing a neurodegenerative or neurological disease or condition in an organism comprising contacting the organism with an siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating or preventing a proliferative disease or condition in an organism comprising contacting the organism with an siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating or preventing a cardiovascular disease or condition in an organism comprising contacting the organism with an siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating or preventing a renal disease or condition in an organism comprising contacting the organism with an siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating or preventing a ocular disease or condition in an organism comprising contacting the organism with an siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating or preventing viral disease or infection in an organism comprising contacting the organism with an siNA molecule of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor gene in the organism.

The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed herein (e.g., cancers and other proliferative conditions, viral infection, inflammatory disease, autoimmunity, respiratory disease, pulmonary disease, cardiovascular disease, neurological disease, renal disease, ocular disease, etc.). For example, to treat a particular disease, condition, trait, genotype or phenotype, the siNA molecules can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.

In another embodiment, the invention features a method of modulating the expression of more than one interleukin (e.g., any IL-1 through IL-27) and/or interleukin receptor (e.g., any IL-1R through IL-27R) genes in an organism comprising contacting the organism with one or more siNA molecules of the invention under conditions suitable to modulate the expression of the interleukin and/or interleukin receptor genes in the organism.

The siNA molecules of the invention can be designed to down regulate or inhibit target (e.g., interleukin and/or interleukin receptor) gene expression through RNAi targeting of a variety of RNA molecules. In one embodiment, the siNA molecules of the invention are used to target various RNAs corresponding to a target gene. Non-limiting examples of such RNAs include messenger RNA (mRNA), alternate RNA splice variants of target gene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates. If alternate splicing produces a family of transcripts that are distinguished by usage of appropriate exons, the instant invention can be used to inhibit gene expression through the appropriate exons to specifically inhibit or to distinguish among the functions of gene family members. For example, a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms. Use of the invention to target the exon containing the transmembrane domain can be used to determine the functional consequences of pharmaceutical targeting of membrane bound as opposed to the secreted form of the protein. Non-limiting examples of applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymorphism mapping with siNA molecules of the invention. Such applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as interleukin and/or interleukin receptor family genes. As such, siNA molecules targeting multiple interleukin and/or interleukin receptor targets can provide increased therapeutic effect. In addition, siNA can be used to characterize pathways of gene function in a variety of applications. For example, the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in, for example, respiratory disease.

In one embodiment, siNA molecule(s) and/or methods of the invention are used to down regulate the expression of gene(s) that encode RNA referred to by Genbank Accession Nos., for example interleukin and/or interleukin receptor genes encoding RNA sequence(s) referred to herein by Genbank Accession number, for example, Genbank Accession Nos. shown in Table I.

In one embodiment, the invention features a method comprising: (a) generating a library of siNA constructs having a predetermined complexity; and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target RNA sequence. In one embodiment, the siNA molecules of (a) have strands of a fixed length, for example, about 23 nucleotides in length. In another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, Northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

In one embodiment, the invention features a method comprising: (a) generating a randomized library of siNA constructs having a predetermined complexity, such as of 4^(N), where N represents the number of base paired nucleotides in each of the siNA construct strands (e.g., for an siNA construct having 21 nucleotide sense and antisense strands with 19 base pairs, the complexity would be 4¹⁹); and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target interleukin and/or interleukin receptor RNA sequence. In another embodiment, the siNA molecules of (a) have strands of a fixed length, for example about 23 nucleotides in length. In yet another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described in Example 6 herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of interleukin and/or interleukin receptor RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, Northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target interleukin and/or interleukin receptor RNA sequence. The target interleukin and/or interleukin receptor RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

In another embodiment, the invention features a method comprising: (a) analyzing the sequence of a RNA target encoded by a target gene; (b) synthesizing one or more sets of siNA molecules having sequence complementary to one or more regions of the RNA of (a); and (c) assaying the siNA molecules of (b) under conditions suitable to determine RNAi targets within the target RNA sequence. In one embodiment, the siNA molecules of (b) have strands of a fixed length, for example about 23 nucleotides in length. In another embodiment, the siNA molecules of (b) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. Fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, Northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by expression in in vivo systems.

By “target site” is meant a sequence within a target RNA that is “targeted” for cleavage mediated by an siNA construct which contains sequences within its antisense region that are complementary to the target sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. Production of cleavage products from 1-5% of the target RNA is sufficient to detect above the background for most methods of detection.

In one embodiment, the invention features a composition comprising an siNA molecule of the invention, which can be chemically-modified, in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a pharmaceutical composition comprising siNA molecules of the invention, which can be chemically-modified, targeting one or more genes in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a method for diagnosing a disease or condition in a subject comprising administering to the subject a composition of the invention under conditions suitable for the diagnosis of the disease or condition in the subject. In another embodiment, the invention features a method for treating or preventing a disease or condition in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease or condition in the subject, alone or in conjunction with one or more other therapeutic compounds. In yet another embodiment, the invention features a method for reducing or preventing, for example, respiratory disease (e.g., asthma) in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the reduction or prevention of the respiratory disease in the subject.

In another embodiment, the invention features a method for validating a interleukin and/or interleukin receptor gene target, comprising: (a) synthesizing an siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a interleukin and/or interleukin receptor target gene; (b) introducing the siNA molecule into a cell, tissue, or organism under conditions suitable for modulating expression of the interleukin and/or interleukin receptor target gene in the cell, tissue, or organism; and (c) determining the function of the gene by assaying for any phenotypic change in the cell, tissue, or organism.

In another embodiment, the invention features a method for validating a interleukin and/or interleukin receptor target comprising: (a) synthesizing an siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a interleukin and/or interleukin receptor target gene; (b) introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the interleukin and/or interleukin receptor target gene in the biological system; and (c) determining the function of the gene by assaying for any phenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurified form, from biological sources, including but not limited to human or animal, wherein the system comprises the components required for RNAi activity. The term “biological system” includes, for example, a cell, tissue, or organism, or extract thereof. The term biological system also includes reconstituted RNAi systems that can be used in an in vitro setting.

By “phenotypic change” is meant any detectable change to a cell that occurs in response to contact or treatment with a nucleic acid molecule of the invention (e.g., siNA). Such detectable changes include, but are not limited to, changes in shape, size, proliferation, motility, protein expression or RNA expression or other physical or chemical changes as can be assayed by methods known in the art. The detectable change can also include expression of reporter genes/molecules such as Green Florescent Protein (GFP) or various tags that are used to identify an expressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing an siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of a interleukin and/or interleukin receptor target gene in a biological system, including, for example, in a cell, tissue, or organism. In another embodiment, the invention features a kit containing more than one siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of more than one interleukin and/or interleukin receptor target gene in a biological system, including, for example, in a cell, tissue, or organism.

In one embodiment, the invention features a cell containing one or more siNA molecules of the invention, which can be chemically-modified. In another embodiment, the cell containing an siNA molecule of the invention is a mammalian cell. In yet another embodiment, the cell containing an siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of an siNA molecule of the invention, which can be chemically-modified, comprises: (a) synthesis of two complementary strands of the siNA molecule; (b) annealing the two complementary strands together under conditions suitable to obtain a double-stranded siNA molecule. In another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing an siNA duplex molecule comprising: (a) synthesizing a first oligonucleotide sequence strand of the siNA molecule, wherein the first oligonucleotide sequence strand comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence strand of the siNA; (b) synthesizing the second oligonucleotide sequence strand of siNA on the scaffold of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence strand further comprises a chemical moiety than can be used to purify the siNA duplex; (c) cleaving the linker molecule of (a) under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex; and (d) purifying the siNA duplex utilizing the chemical moiety of the second oligonucleotide sequence strand.

In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example under hydrolysis conditions using an alkylamine base such as methylamine. In one embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place concomitantly. In another embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group, which can be employed in a trityl-on synthesis strategy as described herein. In yet another embodiment, the chemical moiety, such as a dimethoxytrityl group, is removed during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solution phase synthesis or hybrid phase synthesis wherein both strands of the siNA duplex are synthesized in tandem using a cleavable linker attached to the first sequence which acts a scaffold for synthesis of the second sequence. Cleavage of the linker under conditions suitable for hybridization of the separate siNA sequence strands results in formation of the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizing an siNA duplex molecule comprising: (a) synthesizing one oligonucleotide sequence strand of the siNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of another oligonucleotide sequence; (b) synthesizing a second oligonucleotide sequence having complementarity to the first sequence strand on the scaffold of (a), wherein the second sequence comprises the other strand of the double-stranded siNA molecule and wherein the second sequence further comprises a chemical moiety than can be used to isolate the attached oligonucleotide sequence; (c) purifying the product of (b) utilizing the chemical moiety of the second oligonucleotide sequence strand under conditions suitable for isolating the full-length sequence comprising both siNA oligonucleotide strands connected by the cleavable linker and under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example under hydrolysis conditions. In another embodiment, cleavage of the linker molecule in (c) above takes place after deprotection of the oligonucleotide. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity or differing reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place either concomitantly or sequentially. In one embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group.

In another embodiment, the invention features a method for making a double-stranded siNA molecule in a single synthetic process comprising: (a) synthesizing an oligonucleotide having a first and a second sequence, wherein the first sequence is complementary to the second sequence, and the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and wherein a terminal 5′-protecting group, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains on the oligonucleotide having the second sequence; (b) deprotecting the oligonucleotide whereby the deprotection results in the cleavage of the linker joining the two oligonucleotide sequences; and (c) purifying the product of (b) under conditions suitable for isolating the double-stranded siNA molecule, for example using a trityl-on synthesis strategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of the invention comprises the teachings of Scaringe et al., U.S. Pat. Nos. 5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein in their entirety.

In one embodiment, the invention features siNA constructs that mediate RNAi against interleukin and/or interleukin receptor, wherein the siNA construct comprises one or more chemical modifications, for example, one or more chemical modifications having any of Formulae I-VII or any combination thereof that increases the nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generating siNA molecules with increased nuclease resistance comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into an siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased nuclease resistance.

In one embodiment, the invention features siNA constructs that mediate RNAi against interleukin and/or interleukin receptor, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the sense and antisense strands of the siNA construct.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the sense and antisense strands of the siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into an siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the sense and antisense strands of the siNA molecule.

In one embodiment, the invention features siNA constructs that mediate RNAi against interleukin and/or interleukin receptor, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediate RNAi against interleukin and/or interleukin receptor, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into an siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into an siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediate RNAi against interleukin and/or interleukin receptor, wherein the siNA construct comprises one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA construct.

In another embodiment, the invention features a method for generating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to a chemically-modified siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into an siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA molecule.

In one embodiment, the invention features chemically-modified siNA constructs that mediate RNAi against interleukin and/or interleukin receptor in a cell, wherein the chemical modifications do not significantly effect the interaction of siNA with a target RNA molecule, DNA molecule and/or proteins or other factors that are essential for RNAi in a manner that would decrease the efficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against interleukin and/or interleukin receptor comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into an siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity.

In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against interleukin and/or interleukin receptor target RNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into an siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against interleukin and/or interleukin receptor target DNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into an siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the target DNA.

In one embodiment, the invention features siNA constructs that mediate RNAi against interleukin and/or interleukin receptor, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the cellular uptake of the siNA construct.

In another embodiment, the invention features a method for generating siNA molecules against interleukin and/or interleukin receptor with improved cellular uptake comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into an siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediate RNAi against interleukin and/or interleukin receptor, wherein the siNA construct comprises one or more chemical modifications described herein that increases the bioavailability of the siNA construct, for example, by attaching polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the siNA construct, or by attaching conjugates that target specific tissue types or cell types in vivo. Non-limiting examples of such conjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability, comprising (a) introducing a conjugate into the structure of an siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such conjugates can include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; polyamines, such as spermine or spermidine; and others.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence is chemically modified in a manner that it can no longer act as a guide sequence for efficiently mediating RNA interference and/or be recognized by cellular proteins that facilitate RNAi.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein the second sequence is designed or modified in a manner that prevents its entry into the RNAi pathway as a guide sequence or as a sequence that is complementary to a target nucleic acid (e.g., RNA) sequence. Such design or modifications are expected to enhance the activity of siNA and/or improve the specificity of siNA molecules of the invention. These modifications are also expected to minimize any off-target effects and/or associated toxicity.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence is incapable of acting as a guide sequence for mediating RNA interference.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence does not have a terminal 5′-hydroxyl (5′-OH) or 5′-phosphate group.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5′-end of said second sequence. In one embodiment, the terminal cap moiety comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5′-end and 3′-end of said second sequence. In one embodiment, each terminal cap moiety individually comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved specificity for down regulating or inhibiting the expression of a target nucleic acid (e.g., a DNA or RNA such as a gene or its corresponding RNA), comprising (a) introducing one or more chemical modifications into the structure of an siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved specificity. In another embodiment, the chemical modification used to improve specificity comprises terminal cap modifications at the 5′-end, 3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal cap modifications can comprise, for example, structures shown in FIG. 10 (e.g. inverted deoxyabasic moieties) or any other chemical modification that renders a portion of the siNA molecule (e.g. the sense strand) incapable of mediating RNA interference against an off target nucleic acid sequence. In a non-limiting example, an siNA molecule is designed such that only the antisense sequence of the siNA molecule can serve as a guide sequence for RISC mediated degradation of a corresponding target RNA sequence. This can be accomplished by rendering the sense sequence of the siNA inactive by introducing chemical modifications to the sense strand that preclude recognition of the sense strand as a guide sequence by RNAi machinery. In one embodiment, such chemical modifications comprise any chemical group at the 5′-end of the sense strand of the siNA, or any other group that serves to render the sense strand inactive as a guide sequence for mediating RNA interference. These modifications, for example, can result in a molecule where the 5′-end of the sense strand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphate group (e.g., phosphate, diphosphate, triphosphate, cyclic phosphate etc.). Non-limiting examples of such siNA constructs are described herein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”, and “Stab 24/25” chemistries and variants thereof (see Table IV) wherein the 5′-end and 3′-end of the sense strand of the siNA do not comprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved specificity for down regulating or inhibiting the expression of a target nucleic acid (e.g., a DNA or RNA such as a gene or its corresponding RNA), comprising introducing one or more chemical modifications into the structure of an siNA molecule that prevent a strand or portion of the siNA molecule from acting as a template or guide sequence for RNAi activity. In one embodiment, the inactive strand or sense region of the siNA molecule is the sense strand or sense region of the siNA molecule, i.e. the strand or region of the siNA that does not have complementarity to the target nucleic acid sequence. In one embodiment, such chemical modifications comprise any chemical group at the 5′-end of the sense strand or region of the siNA that does not comprise a 5′-hydroxyl (5′-OH) or 5′-phosphate group, or any other group that serves to render the sense strand or sense region inactive as a guide sequence for mediating RNA interference. Non-limiting examples of such siNA constructs are described herein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”, and “Stab 24/25” chemistries and variants thereof (see Table IV) wherein the 5′-end and 3′-end of the sense strand of the siNA do not comprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for screening siNA molecules that are active in mediating RNA interference against a target nucleic acid sequence comprising (a) generating a plurality of unmodified siNA molecules, (b) screening the siNA molecules of step (a) under conditions suitable for isolating siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence, and (c) introducing chemical modifications (e.g. chemical modifications as described herein or as otherwise known in the art) into the active siNA molecules of (b). In one embodiment, the method further comprises re-screening the chemically modified siNA molecules of step (c) under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence.

In one embodiment, the invention features a method for screening chemically modified siNA molecules that are active in mediating RNA interference against a target nucleic acid sequence comprising (a) generating a plurality of chemically modified siNA molecules (e.g. siNA molecules as described herein or as otherwise known in the art), and (b) screening the siNA molecules of step (a) under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter, that is capable of interacting with another compound, such as a receptor, either directly or indirectly. The receptor that interacts with a ligand can be present on the surface of a cell or can alternately be an intercellular receptor. Interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing an excipient formulation to an siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, nanoparticles, receptors, ligands, and others.

In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing nucleotides having any of Formulae I-VII or any combination thereof into an siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalently attached to siNA compounds of the present invention. The attached PEG can be any molecular weight, preferably from about 2,000 to about 50,000 daltons (Da).

The present invention can be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples and/or subjects. For example, preferred components of the kit include an siNA molecule of the invention and a vehicle that promotes introduction of the siNA into cells of interest as described herein (e.g., using lipids and other methods of transfection known in the art, see for example Beigelman et al, U.S. Pat. No. 6,395,713). The kit can be used for target validation, such as in determining gene function and/or activity, or in drug optimization, and in drug discovery (see for example Usman et al., U.S. Ser. No. 60/402,996). Such a kit can also include instructions to allow a user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interfering RNA”, “siRNA”, “short interfering nucleic acid molecule”, “short interfering oligonucleotide molecule”, or “chemically-modified short interfering nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication, for example by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner; see for example Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples of siNA molecules of the invention are shown in FIGS. 4-6, and Tables II and III herein. For example the siNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double-stranded structure, for example wherein the double-stranded region is about 19 base pairs); the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, the siNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s). The siNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi. The siNA can also comprise a single-stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single-stranded polynucleotide can further comprise a terminal phosphate group, such as a 5′-phosphate (see for example Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certain embodiments, the siNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the siNA molecules of the invention comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the siNA molecule of the invention interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene. As used herein, siNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules of the invention lack 2′-hydroxy (2′-OH) containing nucleotides. Applicant describes in certain embodiments short interfering nucleic acids that do not require the presence of nucleotides having a 2′-hydroxy group for mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2′-OH group). Such siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. The modified short interfering nucleic acid molecules of the invention can also be referred to as short interfering modified oligonucleotides “siMON.” As used herein, the term siNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, siNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

In one embodiment, an siNA molecule of the invention is a duplex forming oligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al., U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and McSwiggen et al., PCT/US04/16390, filed May 24, 2004).

In one embodiment, an siNA molecule of the invention is a multifunctional siNA, (see for example FIGS. 16-22 and Jadhav et al., U.S. Ser. No. 60/543,480 filed Feb. 10, 2004 and McSwiggen et al., PCT/US04/16390, filed May 24, 2004). The multifunctional siNA of the invention can comprise sequence targeting, for example, two regions of interleukin and/or interleukin receptor RNA (see for example target sequences in Tables II and III).

By “asymmetric hairpin” as used herein is meant a linear siNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non-nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex with loop. For example, an asymmetric hairpin siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g. about 19 to about 22, or about 19, 20, 21, or 22 nucleotides) and a loop region comprising about 4 to about 8 (e.g., about 4, 5, 6, 7, or 8) nucleotides, and a sense region having about 3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotides that are complementary to the antisense region. The asymmetric hairpin siNA molecule can also comprise a 5′-terminal phosphate group that can be chemically modified. The loop portion of the asymmetric hairpin siNA molecule can comprise nucleotides, non-nucleotides, linker molecules, or conjugate molecules as described herein.

By “asymmetric duplex” as used herein is meant an siNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex. For example, an asymmetric duplex siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system e.g. about 19 to about 22 (e.g. about 19, 20, 21, or 22) nucleotides and a sense region having about 3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotides that are complementary to the antisense region.

By “modulate” is meant that the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator. For example, the term “modulate” can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention. In one embodiment, inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule. In another embodiment, inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches. In another embodiment, inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.

By “gene”, or “target gene”, is meant, a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. A gene or target gene can also encode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Such non-coding RNAs can serve as target nucleic acid molecules for siNA mediated RNA interference in modulating the activity of fRNA or ncRNA involved in functional or regulatory cellular processes. Aberrant fRNA or ncRNA activity leading to disease can therefore be modulated by siNA molecules of the invention. siNA molecules targeting fRNA and ncRNA can also be used to manipulate or alter the genotype or phenotype of an organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing (e.g., transamination, methylation etc.). The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts. For a review, see for example Snyder and Gerstein, 2003, Science, 300, 258-260.

By “non-canonical base pair” is meant any non-Watson Crick base pair, such as mismatches and/or wobble base pairs, including flipped mismatches, single hydrogen bond mismatches, trans-type mismatches, triple base interactions, and quadruple base interactions. Non-limiting examples of such non-canonical base pairs include, but are not limited to, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AA N7 amino, CC 2-carbonyl-amino(H1)-N-3-amino(H2), GA sheared, UC 4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AU reverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AA N1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC 4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H—N3, GA carbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A) N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonyl amino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

By “interleukin” is meant, any interleukin (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, and IL-27) polypeptide, protein and/or a polynucleotide encoding an interleukin protein, peptide, or portion thereof (such as polynucleotides referred to by Genbank Accession numbers in Table I or any other interleukin transcript derived from an interleukin gene). The term “interleukin” is also meant to include other interleukin encoding sequence, such as mutant interleukin genes, splice variants of interleukin genes, and interleukin gene polymorphisms, such as those associated with a disease, trait, or condition.

By “interleukin protein” is meant, any interleukin peptide or protein or a component thereof, wherein the peptide or protein is encoded by an interleukin gene or having interleukin activity.

By “interleukin receptor” is meant, any interleukin receptor (e.g., IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, IL-16R, IL-17R, IL-18R, IL-19R, IL-20R, IL-21R, IL-22R, IL-23R, IL-24R, IL-25R, IL-26R, and IL-27R) polypeptide, protein and/or a polynucleotide encoding an interleukin receptor protein, peptide, or portion thereof (such as polynucleotides referred to by Genbank Accession numbers in Table I or any other interleukin receptor transcript derived from an interleukin receptor gene). The term “interleukin receptor” is also meant to include other interleukin receptor encoding sequence, such as mutant interleukin receptor genes, splice variants of interleukin receptor genes, and interleukin receptor gene polymorphisms, such as those associated with a disease, trait, or condition.

By “interleukin receptor protein” is meant, any interleukin receptor peptide or protein or a component thereof, wherein the peptide or protein is encoded by an interleukin receptor gene or having interleukin receptor activity.

By “homologous sequence” is meant, a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors. A homologous sequence can be a nucleotide sequence that is shared by two or more non-coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect homology (e.g., 100%), as partially homologous sequences are also contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system or organism to another biological system or organism. The polynucleotide can include both coding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of an siNA molecule having complementarity to an antisense region of the siNA molecule. In addition, the sense region of an siNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of an siNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of an siNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the siNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.

In one embodiment, siNA molecules of the invention that down regulate or reduce interleukin and/or interleukin receptor gene expression are used for preventing or reducing cancers and other proliferative conditions, viral infection, inflammatory disease, autoimmunity, respiratory disease, pulmonary disease, cardiovascular disease, neurological disease, renal disease, ocular disease, liver disease, mitochondrial disease, endocrine disease, prion disease, reproduction related diseases and conditions or any other disease associated with interleukin and/or interleukin receptor gene expression in a subject. In one embodiment, the siNA molecules of the invention that down regulate or reduce interleukin and/or interleukin receptor gene expression are used for treating or preventing asthma, chronic obstructive pulmonary disease or “COPD”, allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation, allergies, impeded respiration, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, or emphysema in a subject.

By “cancer” is meant a group of diseases characterized by uncontrolled growth and spread of abnormal cells.

By “proliferative disease” or “cancer” is meant, any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art; including AIDS related cancers such as Kaposi's sarcoma; breast cancers; bone cancers such as Osteosarcoma, Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors, Adamantinomas, and Chordomas; Brain cancers such as Meningiomas, Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas, and Metastatic brain cancers; cancers of the head and neck including various lymphomas such as mantle cell lymphoma, non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder and bile duct cancers, cancers of the retina such as retinoblastoma, cancers of the esophagus, gastric cancers, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, colorectal cancer, lung cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervical cancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrug resistant cancers; and proliferative diseases and conditions, such as neovascularization associated with tumor angiogenesis, macular degeneration (e.g., wet/dry AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration and other proliferative diseases and conditions such as restenosis and polycystic kidney disease, and any other cancer or proliferative disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a cell or tissue, alone or in combination with other therapies.

By “inflammatory disease” or “inflammatory condition” is meant any disease, condition, trait, genotype or phenotype characterized by an inflammatory or allergic process as is known in the art, such as inflammation, acute inflammation, chronic inflammation, atherosclerosis, restenosis, asthma, allergic rhinitis, atopic dermatitis, psoriasis, septic shock, rheumatoid arthritis, inflammatory bowl disease, inflammatory pelvic disease, pain, ocular inflammatory disease, celiac disease, Leigh Syndrome, Glycerol Kinase Deficiency, Familial eosinophilia (FE), autosomal recessive spastic ataxia, laryngeal inflammatory disease; Tuberculosis, Chronic cholecystitis, Bronchiectasis, Silicosis and other pneumoconiosis, and any other inflammatory disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a cell or tissue, alone or in combination with other therapies.

By “respiratory disease” is meant, any disease or condition affecting the respiratory tract, such as asthma, chronic obstructive pulmonary disease or “COPD”, allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation, allergies, impeded respiration, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, and any other respiratory disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a cell or tissue, alone or in combination with other therapies.

By “autoimmune disease” or “autoimmune condition” is meant, any disease, condition, trait, genotype or phenotype characterized by autoimmunity as is known in the art, such as multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderms, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Menier's syndrome; transplantation rejection (e.g., prevention of allograft rejection) pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease, and any other autoimmune disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a cell or tissue, alone or in combination with other therapies.

By “neurological disease” or “neurological disease” is meant any disease, disorder, or condition affecting the central or peripheral nervous system, including ADHD, AIDS—Neurological Complications, Absence of the Septum Pellucidum, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation, Aspartame, Asperger Syndrome, Ataxia Telangiectasia, Ataxia, Attention Deficit-Hyperactivity Disorder, Autism, Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease, Behcet's Disease, Bell's Palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy, Benign Intracranial Hypertension, Bernhardt-Roth Syndrome, Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, Brain Aneurysm, Brain Injury, Brain and Spinal Tumors, Brown-Sequard Syndrome, Bulbospinal Muscular Atrophy, Canavan Disease, Carpal Tunnel Syndrome, Causalgia, Cavernomas, Cavernous Angioma, Cavernous Malformation, Central Cervical Cord Syndrome, Central Cord Syndrome, Central Pain Syndrome, Cephalic Disorders, Cerebellar Degeneration, Cerebellar Hypoplasia, Cerebral Aneurysm, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome, Charcot-Marie-Tooth Disorder, Chiari Malformation, Chorea, Choreoacanthocytosis, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Coma, including Persistent Vegetative State, Complex Regional Pain Syndrome, Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy, Congenital Vascular Cavernous Malformations, Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis, Creutzfeldt-Jakob Disease, Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalic Inclusion Body Disease (CIBD), Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier's Syndrome, Dejerine-Klumpke Palsy, Dementia—Multi-Infarct, Dementia—Subcortical, Dementia With Lewy Bodies, Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Dravet's Syndrome, Dysautonomia, Dysgraphia, Dyslexia, Dysphagia, Dyspraxia, Dystonias, Early Infantile Epileptic Encephalopathy, Empty Sella Syndrome, Encephalitis Lethargica, Encephalitis and Meningitis, Encephaloceles, Encephalopathy, Encephalotrigeminal Angiomatosis, Epilepsy, Erb's Palsy, Erb-Duchenne and Dejerine-Klumpke Palsies, Fabry's Disease, Fahr's Syndrome, Fainting, Familial Dysautonomia, Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification, Familial Spastic Paralysis, Febrile Seizures (e.g., GEFS and GEFS plus), Fisher Syndrome, Floppy Infant Syndrome, Friedreich's Ataxia, Gaucher's Disease, Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease, Giant Cell Arteritis, Giant Cell Inclusion Disease, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia, Guillain-Barre Syndrome, HTLV-1 Associated Myelopathy, Hallervorden-Spatz Disease, Head Injury, Headache, Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans, Hereditary Neuropathies, Hereditary Spastic Paraplegia, Heredopathia Atactica Polyneuritiformis, Herpes Zoster Oticus, Herpes Zoster, Hirayama Syndrome, Holoprosencephaly, Huntington's Disease, Hydranencephaly, Hydrocephalus—Normal Pressure, Hydrocephalus, Hydromyelia, Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis, Incontinentia Pigmenti, Infantile Hypotonia, Infantile Phytanic Acid Storage Disease, Infantile Refsum Disease, Infantile Spasms, Inflammatory Myopathy, Intestinal Lipodystrophy, Intracranial Cysts, Intracranial Hypertension, Isaac's Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome, Kleine-Levin syndrome, Klippel Feil Syndrome, Klippel-Trenaunay Syndrome (KTS), Kluver-Bucy Syndrome, Korsakoff's Amnesic Syndrome, Krabbe Disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-Critchley Syndrome, Lewy Body Dementia, Lissencephaly, Locked-In Syndrome, Lou Gehrig's Disease, Lupus—Neurological Sequelae, Lyme Disease—Neurological Complications, Machado-Joseph Disease, Macrencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome, Meningitis, Menkes Disease, Meralgia Paresthetica, Metachromatic Leukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome, Mini-Strokes, Mitochondrial Myopathies, Mobius Syndrome, Monomelic Amyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses, Mucopolysaccharidoses, Multi-Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy with Orthostatic Hypotension, Multiple System Atrophy, Muscular Dystrophy, Myasthenia—Congenital, Myasthenia Gravis, Myelinoclastic Diffuse Sclerosis, Myoclonic Encephalopathy of Infants, Myoclonus, Myopathy—Congenital, Myopathy—Thyrotoxic, Myopathy, Myotonia Congenita, Myotonia, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with Brain Iron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome, Neurological Complications of AIDS, Neurological Manifestations of Pompe Disease, Neuromyelitis Optica, Neuromyotonia, Neuronal Ceroid Lipofuscinosis, Neuronal Migration Disorders, Neuropathy—Hereditary, Neurosarcoidosis, Neurotoxicity, Nevus Cavemosus, Niemann-Pick Disease, O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Occult Spinal Dysraphism Sequence, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome, Pain—Chronic, Paraneoplastic Syndromes, Paresthesia, Parkinson's Disease, Parmyotonia Congenita, Paroxysmal Choreoathetosis, Paroxysmal Hemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir II Syndrome, Perineural Cysts, Periodic Paralyses, Peripheral Neuropathy, Periventricular Leukomalacia, Persistent Vegetative State, Pervasive Developmental Disorders, Phytanic Acid Storage Disease, Pick's Disease, Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease, Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia, Postinfectious Encephalomyelitis, Postural Hypotension, Postural Orthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, Primary Lateral Sclerosis, Prion Diseases, Progressive Hemifacial Atrophy, Progressive Locomotor Ataxia, Progressive Multifocal Leukoencephalopathy, Progressive Sclerosing Poliodystrophy, Progressive Supranuclear Palsy, Pseudotumor Cerebri, Pyridoxine Dependent and Pyridoxine Responsive Siezure Disorders, Ramsay Hunt Syndrome Type I, Ramsay Hunt Syndrome Type II, Rasmussen's Encephalitis and other autoimmune epilepsies, Reflex Sympathetic Dystrophy Syndrome, Refsum Disease—Infantile, Refsum Disease, Repetitive Motion Disorders, Repetitive Stress Injuries, Restless Legs Syndrome, Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome, Riley-Day Syndrome, SUNCT Headache, Sacral Nerve Root Cysts, Saint Vitus Dance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease, Schizencephaly, Seizure Disorders, Septo-Optic Dysplasia, Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles, Shy-Drager Syndrome, Sjogren's Syndrome, Sleep Apnea, Sleeping Sickness, Soto's Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction, Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy, Spinocerebellar Atrophy, Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, Subacute Sclerosing Panencephalitis, Subcortical Arteriosclerotic Encephalopathy, Swallowing Disorders, Sydenham Chorea, Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia, Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, Tarlov Cysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal Cord Syndrome, Thomsen Disease, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, Transient Ischemic Attack, Transmissible Spongiform Encephalopathies, Transverse Myelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, Tropical Spastic Paraparesis, Tuberous Sclerosis, Vascular Erectile Tumor, Vasculitis including Temporal Arteritis, Von Economo's Disease, Von Hippel-Lindau disease (VHL), Von Recklinghausen's Disease, Wallenberg's Syndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, West Syndrome, Whipple's Disease, Williams Syndrome, Wilson's Disease, X-Linked Spinal and Bulbar Muscular Atrophy, and Zellweger Syndrome.

By “infectious disease” is meant any disease, condition, trait, genotype or phenotype associated with an infectious agent, such as a virus, bacteria, fungus, prion, or parasite. Non-limiting examples of various viral genes that can be targeted using siNA molecules of the invention include Hepatitis C Virus (HCV, for example Genbank Accession Nos: D11168, D50483.1, L38318 and S82227), Hepatitis B Virus (HBV, for example GenBank Accession No. AF100308.1), Human Immunodeficiency Virus type 1 (HIV-1, for example GenBank Accession No. U51188), Human Immunodeficiency Virus type 2 (HIV-2, for example GenBank Accession No. X60667), West Nile Virus (WNV for example GenBank accession No. NC_(—)001563), cytomegalovirus (CMV for example GenBank Accession No. NC_(—)001347), respiratory syncytial virus (RSV for example GenBank Accession No. NC_(—)001781), influenza virus (for example GenBank Accession No. AF037412, rhinovirus (for example, GenBank accession numbers: D00239, X02316, X01087, L24917, M16248, K02121, X01087), papillomavirus (for example GenBank Accession No. NC_(—)001353), Herpes Simplex Virus (HSV for example GenBank Accession No. NC_(—)001345), and other viruses such as HTLV (for example GenBank Accession No. AJ430-458). Due to the high sequence variability of many viral genomes, selection of siNA molecules for broad therapeutic applications would likely involve the conserved regions of the viral genome. Nonlimiting examples of conserved regions of the viral genomes include but are not limited to 5′-Non Coding Regions (NCR), 3′-Non Coding Regions (NCR) and/or internal ribosome entry sites (IRES). siNA molecules designed against conserved regions of various viral genomes will enable efficient inhibition of viral replication in diverse patient populations and may ensure the effectiveness of the siNA molecules against viral quasi species which evolve due to mutations in the non-conserved regions of the viral genome. Non-limiting examples of bacterial infections include Actinomycosis, Anthrax, Aspergillosis, Bacteremia, Bacterial Infections and Mycoses, Bartonella Infections, Botulism, Brucellosis, Burkholderia Infections, Campylobacter Infections, Candidiasis, Cat-Scratch Disease, Chlamydia Infections, Cholera, Clostridium Infections, Coccidioidomycosis, Cross Infection, Cryptococcosis, Dermatomycoses, Dermatomycoses, Diphtheria, Ehrlichiosis, Escherichia coli Infections, Fasciitis, Necrotizing, Fusobacterium Infections, Gas Gangrene, Gram-Negative Bacterial Infections, Gram-Positive Bacterial Infections, Histoplasmosis, Impetigo, Klebsiella Infections, Legionellosis, Leprosy, Leptospirosis, Listeria Infections, Lyme Disease, Maduromycosis, Melioidosis, Mycobacterium Infections, Mycoplasma Infections, Mycoses, Nocardia Infections, Onychomycosis, Ornithosis, Plague, Pneumococcal Infections, Pseudomonas Infections, Q Fever, Rat-Bite Fever, Relapsing Fever, Rheumatic Fever, Rickettsia Infections, Rocky Mountain Spotted Fever, Salmonella Infections, Scarlet Fever, Scrub Typhus, Sepsis, Sexually Transmitted Diseases—Bacterial, Bacterial Skin Diseases, Staphylococcal Infections, Streptococcal Infections, Tetanus, Tick-Borne Diseases, Tuberculosis, Tularemia, Typhoid Fever, Typhus, Epidemic Louse-Borne, Vibrio Infections, Yaws, Yersinia Infections, Zoonoses, and Zygomycosis. Non-limiting examples of fungal infections include Aspergillosis, Blastomycosis, Coccidioidomycosis, Cryptococcosis, Fungal Infections of Fingernails and Toenails, Fungal Sinusitis, Histoplasmosis, Histoplasmosis, Mucormycosis, Nail Fungal Infection, Paracoccidioidomycosis, Sporotrichosis, Valley Fever (Coccidioidomycosis), and Mold Allergy.

By “ocular disease” is meant, any disease, condition, trait, genotype or phenotype of the eye and related structures, such as Cystoid Macular Edema, Asteroid Hyalosis, Pathological Myopia and Posterior Staphyloma, Toxocariasis (Ocular Larva Migrans), Retinal Vein Occlusion, Posterior Vitreous Detachment, Tractional Retinal Tears, Epiretinal Membrane, Diabetic Retinopathy, Lattice Degeneration, Retinal Vein Occlusion, Retinal Artery Occlusion, Macular Degeneration (e.g., age related macular degeneration such as wet AMD or dry AMD), Toxoplasmosis, Choroidal Melanoma, Acquired Retinoschisis, Hollenhorst Plaque, Idiopathic Central Serous Chorioretinopathy, Macular Hole, Presumed Ocular Histoplasmosis Syndrome, Retinal Macroaneursym, Retinitis Pigmentosa, Retinal Detachment, Hypertensive Retinopathy, Retinal Pigment Epithelium (RPE) Detachment, Papillophlebitis, Ocular Ischemic Syndrome, Coats' Disease, Leber's Miliary Aneurysm, Conjunctival Neoplasms, Allergic Conjunctivitis, Vernal Conjunctivitis, Acute Bacterial Conjunctivitis, Allergic Conjunctivitis & Vernal Keratoconjunctivitis, Viral Conjunctivitis, Bacterial Conjunctivitis, Chlamydial & Gonococcal Conjunctivitis, Conjunctival Laceration, Episcleritis, Scleritis, Pingueculitis, Pterygium, Superior Limbic Keratoconjunctivitis (SLK of Theodore), Toxic Conjunctivitis, Conjunctivitis with Pseudomembrane, Giant Papillary Conjunctivitis, Terrien's Marginal Degeneration, Acanthamoeba Keratitis, Fungal Keratitis, Filamentary Keratitis, Bacterial Keratitis, Keratitis Sicca/Dry Eye Syndrome, Bacterial Keratitis, Herpes Simplex Keratitis, Sterile Corneal Infiltrates, Phlyctenulosis, Corneal Abrasion & Recurrent Corneal Erosion, Corneal Foreign Body, Chemical Burs, Epithelial Basement Membrane Dystrophy (EBMD), Thygeson's Superficial Punctate Keratopathy, Corneal Laceration, Salzmann's Nodular Degeneration, Fuchs' Endothelial Dystrophy, Crystalline Lens Subluxation, Ciliary-Block Glaucoma, Primary Open-Angle Glaucoma, Pigment Dispersion Syndrome and Pigmentary Glaucoma, Pseudoexfoliation Syndrom and Pseudoexfoliative Glaucoma, Anterior Uveitis, Primary Open Angle Glaucoma, Uveitic Glaucoma & Glaucomatocyclitic Crisis, Pigment Dispersion Syndrome & Pigmentary Glaucoma, Acute Angle Closure Glaucoma, Anterior Uveitis, Hyphema, Angle Recession Glaucoma, Lens Induced Glaucoma, Pseudoexfoliation Syndrome and Pseudoexfoliative Glaucoma, Axenfeld-Rieger Syndrome, Neovascular Glaucoma, Pars Planitis, Choroidal Rupture, Duane's Retraction Syndrome, Toxic/Nutritional Optic Neuropathy, Aberrant Regeneration of Cranial Nerve III, Intracranial Mass Lesions, Carotid-Cavernous Sinus Fistula, Anterior Ischemic Optic Neuropathy, Optic Disc Edema & Papilledema, Cranial Nerve III Palsy, Cranial Nerve IV Palsy, Cranial Nerve VI Palsy, Cranial Nerve VII (Facial Nerve) Palsy, Horner's Syndrome, Internuclear Opthalmoplegia, Optic Nerve Head Hypoplasia, Optic Pit, Tonic Pupil, Optic Nerve Head Drusen, Demyelinating Optic Neuropathy (Optic Neuritis, Retrobulbar Optic Neuritis), Amaurosis Fugax and Transient Ischemic Attack, Pseudotumor Cerebri, Pituitary Adenoma, Molluscum Contagiosum, Canaliculitis, Verruca and Papilloma, Pediculosis and Pthiriasis, Blepharitis, Hordeolum, Preseptal Cellulitis, Chalazion, Basal Cell Carcinoma, Herpes Zoster Ophthalmicus, Pediculosis & Phthiriasis, Blow-out Fracture, Chronic Epiphora, Dacryocystitis, Herpes Simplex Blepharitis, Orbital Cellulitis, Senile Entropion, and Squamous Cell Carcinoma.

By “cardiovascular disease” is meant and disease or condition affecting the heart and vasculature, including but not limited to, coronary heart disease (CHD), cerebrovascular disease (CVD), aortic stenosis, peripheral vascular disease, atherosclerosis, arteriosclerosis, myocardial infarction (heart attack), cerebrovascular diseases (stroke), transient ischaemic attacks (TIA), angina (stable and unstable), atrial fibrillation, arrhythmia, valvular disease, and/or congestive heart failure.

In one embodiment of the present invention, each sequence of an siNA molecule of the invention is independently about 18 to about 24 nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. In another embodiment, the siNA duplexes of the invention independently comprise about 17 to about 23 base pairs (e.g., about 17, 18, 19, 20, 21, 22, or 23). In yet another embodiment, siNA molecules of the invention comprising hairpin or circular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50 or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38, 39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 16 to about 22 (e.g., about 16, 17, 18, 19, 20, 21 or 22) base pairs. Exemplary siNA molecules of the invention are shown in Table II. Exemplary synthetic siNA molecules of the invention are shown in Table III and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through direct dermal application, transdermal application, or injection, with or without their incorporation in biopolymers. In particular embodiments, the nucleic acid molecules of the invention comprise sequences shown in Tables II-III and/or FIGS. 4-5. Examples of such nucleic acid molecules consist essentially of sequences defined in these tables and figures. Furthermore, the chemically modified constructs described in Table IV can be applied to any siNA sequence of the invention.

In another aspect, the invention provides mammalian cells containing one or more siNA molecules of this invention. The one or more siNA molecules can independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to an organism to which the nucleic acid molecules of the invention can be administered. A subject can be a mammal or mammalian cells, including a human or human cells.

The term “phosphorothioate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise a sulfur atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise an acetyl or protected acetyl group.

The term “thiophosphonoacetate” as used herein refers to an internucleotide linkage having Formula I, wherein Z comprises an acetyl or protected acetyl group and W comprises a sulfur atom or alternately W comprises an acetyl or protected acetyl group and Z comprises a sulfur atom.

The term “universal base” as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotide having an acyclic ribose sugar.

The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to for preventing or treating cancers and other proliferative conditions, viral infection, inflammatory disease, autoimmunity, respiratory disease, pulmonary disease, cardiovascular disease, neurological disease, renal disease, ocular disease, liver disease, mitochondrial disease, endocrine disease, prion disease, or reproduction related diseases and conditions in a subject or organism. In one embodiment, siNA molecules of the invention are used in combination with anti-inflammatory agents or bronchodilators as are known in the art to treat or prevent inflammatory and respiratory diseases and/or conditions in a subject or organism.

For example, the siNA molecules can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs (e.g., statins, hypertensive agents etc.) under conditions suitable for the treatment.

In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention, in a manner which allows expression of the siNA molecule. For example, the vector can contain sequence(s) encoding both strands of an siNA molecule comprising a duplex. The vector can also contain sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms an siNA molecule. Non-limiting examples of such expression vectors are described in Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi: 10.1038/nm725.

In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the invention comprises a sequence for an siNA molecule having complementarity to a RNA molecule referred to by a Genbank Accession numbers, for example Genbank Accession Nos. shown in Table I.

In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more siNA molecules, which can be the same or different.

In another aspect of the invention, siNA molecules that interact with target RNA molecules and down-regulate gene encoding target RNA molecules (for example target RNA molecules referred to by Genbank Accession numbers herein) are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecules bind and down-regulate gene function or expression via RNA interference (RNAi). Delivery of siNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis of siNA molecules. The complementary siNA sequence strands, strand 1 and strand 2, are synthesized in tandem and are connected by a cleavable linkage, such as a nucleotide succinate or abasic succinate, which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support. The synthesis can be either solid phase or solution phase, in the example shown, the synthesis is a solid phase synthesis. The synthesis is performed such that a protecting group, such as a dimethoxytrityl group, remains intact on the terminal nucleotide of the tandem oligonucleotide. Upon cleavage and deprotection of the oligonucleotide, the two siNA strands spontaneously hybridize to form an siNA duplex, which allows the purification of the duplex by utilizing the properties of the terminal protecting group, for example by applying a trityl on purification method wherein only duplexes/oligonucleotides with the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplex synthesized by a method of the invention. The two peaks shown correspond to the predicted mass of the separate siNA sequence strands. This result demonstrates that the siNA duplex generated from tandem synthesis can be purified as a single entity using a simple trityl-on purification methodology.

FIG. 3 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi. Double-stranded RNA (dsRNA), which is generated by RNA-dependent RNA polymerase (RdRP) from foreign single-stranded RNA, for example viral, transposon, or other exogenous RNA, activates the DICER enzyme that in turn generates siNA duplexes. Alternately, synthetic or expressed siNA can be introduced directly into a cell by appropriate means. An active siNA complex forms which recognizes a target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or in the synthesis of additional RNA by RNA-dependent RNA polymerase (RdRP), which can activate DICER and result in additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically-modified siNA constructs of the present invention. In the figure, N stands for any nucleotide (adenosine, guanosine, cytosine, uridine, or optionally thymidine, for example thymidine can be substituted in the overhanging regions designated by parenthesis (N N). Various modifications are shown for the sense and antisense strands of the siNA constructs. The antisense strand of constructs A-F comprise sequence complementary to any target nucleic acid sequence of the invention. Furthermore, when a glyceryl moiety (L) is present at the 3′-end of the antisense strand for any construct shown in FIG. 4 A-F, the modified internucleotide linkage is optional.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and having one 3′-terminal phosphorothioate internucleotide linkage and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-deoxy nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 5A-F shows non-limiting examples of specific chemically-modified siNA sequences of the invention. A-F applies the chemical modifications described in FIG. 4A-F to an IL-4R siNA sequence. Such chemical modifications can be applied to any interleukin and/or interleukin receptor sequence and/or interleukin and/or interleukin receptor polymorphism sequence.

FIG. 6 shows non-limiting examples of different siNA constructs of the invention. The examples shown (constructs 1, 2, and 3) have 19 representative base pairs; however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example comprising about 1, 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for RNAi activity. Construct 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and/or in vitro. In another example, construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siNA construct 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA construct 1 in vivo and/or in vitro. As such, the stability and/or activity of the siNA constructs can be modulated based on the design of the siNA construct for use in vivo or in vitro and/or in vitro.

FIG. 7A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1) sequence followed by a region having sequence identical (sense region of siNA) to a predetermined interleukin and/or interleukin receptor target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, which is followed by a loop sequence of defined sequence (X), comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence that will result in an siNA transcript having specificity for a interleukin and/or interleukin receptor target sequence and having self-complementary sense and antisense regions.

FIG. 7C: The construct is heated (for example to about 95° C.) to linearize the sequence, thus allowing extension of a complementary second DNA strand using a primer to the 3′-restriction sequence of the first strand. The double-stranded DNA is then inserted into an appropriate vector for expression in cells. The construct can be designed such that a 3′-terminal nucleotide overhang results from the transcription, for example by engineering restriction sites and/or utilizing a poly-U termination region as described in Paul et al., 2002, Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate double-stranded siNA constructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) site sequence followed by a region having sequence identical (sense region of siNA) to a predetermined interleukin and/or interleukin receptor target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, and which is followed by a 3′-restriction site (R2) which is adjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific to R1 and R2 to generate a double-stranded DNA which is then inserted into an appropriate vector for expression in cells. The transcription cassette is designed such that a U6 promoter region flanks each side of the dsDNA which generates the separate sense and antisense strands of the siNA. Poly T termination sequences can be added to the constructs to generate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determine target sites for siNA mediated RNAi within a particular target nucleic acid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein the antisense region of the siNA constructs has complementarity to target sites across the target nucleic acid sequence, and wherein the sense region comprises sequence complementary to the antisense region of the siNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted into vectors such that (FIG. 9C) transfection of a vector into cells results in the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associated with modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced to identify efficacious target sites within the target nucleic acid sequence.

FIG. 10 shows non-limiting examples of different stabilization chemistries (1-10) that can be used, for example, to stabilize the 3′-end of siNA sequences of the invention, including (1) [3-3′]-inverted deoxyribose; (2) deoxyribonucleotide; (3) [5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5) [5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7) [3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9) [5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. In addition to modified and unmodified backbone chemistries indicated in the figure, these chemistries can be combined with different backbone modifications as described herein, for example, backbone modifications having Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to the terminal modifications shown can be another modified or unmodified nucleotide or non-nucleotide described herein, for example modifications having any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identify chemically modified siNA constructs of the invention that are nuclease resistance while preserving the ability to mediate RNAi activity. Chemical modifications are introduced into the siNA construct based on educated design parameters (e.g. introducing 2′-modifications, base modifications, backbone modifications, terminal cap modifications etc). The modified construct in tested in an appropriate system (e.g. human serum for nuclease resistance, shown, or an animal model for PK/delivery parameters). In parallel, the siNA construct is tested for RNAi activity, for example in a cell culture system such as a luciferase reporter assay). Lead siNA constructs are then identified which possess a particular characteristic while maintaining RNAi activity, and can be further modified and assayed once again. This same approach can be used to identify siNA-conjugate molecules with improved pharmacokinetic profiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules of the invention, including linear and duplex constructs and asymmetric derivatives thereof.

FIG. 13 shows non-limiting examples of chemically modified terminal phosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design self complementary DFO constructs utilizing palindrome and/or repeat nucleic acid sequences that are identified in a target nucleic acid sequence. (i) A palindrome or repeat sequence is identified in a nucleic acid target sequence. (ii) A sequence is designed that is complementary to the target nucleic acid sequence and the palindrome sequence. (iii) An inverse repeat sequence of the non-palindrome/repeat portion of the complementary sequence is appended to the 3′-end of the complementary sequence to generate a self complementary DFO molecule comprising sequence complementary to the nucleic acid target. (iv) The DFO molecule can self-assemble to form a double-stranded oligonucleotide. FIG. 14B shows a non-limiting representative example of a duplex forming oligonucleotide sequence. FIG. 14C shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence. FIG. 14D shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence followed by interaction with a target nucleic acid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementary DFO constructs utilizing palindrome and/or repeat nucleic acid sequences that are incorporated into the DFO constructs that have sequence complementary to any target nucleic acid sequence of interest. Incorporation of these palindrome/repeat sequences allow the design of DFO constructs that form duplexes in which each strand is capable of mediating modulation of target gene expression, for example by RNAi. First, the target sequence is identified. A complementary sequence is then generated in which nucleotide or non-nucleotide modifications (shown as X or Y) are introduced into the complementary sequence that generate an artificial palindrome (shown as XYXYXY in the Figure). An inverse repeat of the non-palindrome/repeat complementary sequence is appended to the 3′-end of the complementary sequence to generate a self complementary DFO comprising sequence complementary to the nucleic acid target. The DFO can self-assemble to form a double-stranded oligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences. FIG. 16A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 3′-ends of each polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 16B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5′-ends of each polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences. FIG. 17A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the second complementary region is situated at the 3′-end of the polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 17B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first complementary region is situated at the 5′-end of the polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional siNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifunctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences. FIG. 18A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 3′-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 18B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5′-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional siNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifunctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences. FIG. 19A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the second complementary region is situated at the 3′-end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 19B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first complementary region is situated at the 5′-end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid molecules, such as separate RNA molecules encoding differing proteins, for example a cytokine and its corresponding receptor, differing viral strains, a virus and a cellular protein involved in viral infection or replication, or differing proteins involved in a common or divergent biologic pathway that is implicated in the maintenance of progression of disease. Each strand of the multifunctional siNA construct comprises a region having complementarity to separate target nucleic acid molecules. The multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target. These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.

FIG. 21 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid sequences within the same target nucleic acid molecule, such as alternate coding regions of a RNA, coding and non-coding regions of a RNA, or alternate splice variant regions of a RNA. Each strand of the multifunctional siNA construct comprises a region having complementarity to the separate regions of the target nucleic acid molecule. The multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target region. These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.

FIG. 22 shows a non-limiting example of reduction of IL-4R mRNA in HeLa cells mediated by siNAs that target IL-4R mRNA. HeLa cells were transfected with 0.25 ug/well of lipid complexed with 25 nM siNA. Active siNA constructs comprising Stab 9/22 stabilization chemistry were compared to matched chemistry irrelevant siNA control constructs (IC), and cells transfected with lipid alone (transfection control). As shown in the figure, the siNA constructs significantly reduce IL-4R RNA expression.

DETAILED DESCRIPTION OF THE INVENTION Mechanism of Action of Nucleic Acid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNA interference mediated by short interfering RNA as is presently known, and is not meant to be limiting and is not an admission of prior art. Applicant demonstrates herein that chemically-modified short interfering nucleic acids possess similar or improved capacity to mediate RNAi as do siRNA molecules and are expected to possess improved stability and activity in vivo; therefore, this discussion is not meant to be limiting only to siRNA and can be applied to siNA as a whole. By “improved capacity to mediate RNAi” or “improved RNAi activity” is meant to include RNAi activity measured in vitro and/or in vivo where the RNAi activity is a reflection of both the ability of the siNA to mediate RNAi and the stability of the siNAs of the invention. In this invention, the product of these activities can be increased in vitro and/or in vivo compared to an all RNA siRNA or an siNA containing a plurality of ribonucleotides. In some cases, the activity or stability of the siNA molecule can be decreased (i.e., less than ten-fold), but the overall activity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as Dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from Dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex containing an siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188). In addition, RNA interference can also involve small RNA (e.g., micro-RNA or miRNA) mediated gene silencing, presumably though cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see for example Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237). As such, siNA molecules of the invention can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two 2-nucleotide 3′-terminal nucleotide overhangs. Furthermore, substitution of one or both siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3′-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of an siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNA molecules lacking a 5′-phosphate are active when introduced exogenously, suggesting that 5′-phosphorylation of siRNA constructs may occur in vivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (“small” refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. All of these references are incorporated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45 second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoro nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aqueous methylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.

The method of synthesis used for RNA including certain siNA molecules of the invention follows the procedure as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2′-O-methylated nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA·3HF to provide a 1.4 M HF concentration) and heated to 65° C. After 1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO:1/1 (0.8 mL) at 65° C. for 15 minutes. The vial is brought to room temperature TEA·3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 minutes. The sample is cooled at −20° C. and then quenched with 1.5 M NH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃ solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 minutes. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandem synthesis methodology as described in Example 1 herein, wherein both siNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siNA fragments or strands that hybridize and permit purification of the siNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms. The tandem synthesis of siNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.

An siNA molecule can also be assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water.

In another aspect of the invention, siNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al., supra; all of which are incorporated by reference herein). All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.

There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the siNA nucleic acid molecules of the instant invention so long as the ability of siNA to promote RNAi is cells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonate linkages improves stability, excessive modifications can cause some toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, resulting in increased efficacy and higher specificity of these molecules.

Short interfering nucleic acid (siNA) molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Accordingly, the in vitro and/or in vivo activity should not be significantly lowered. In cases in which modulation is the goal, therapeutic nucleic acid molecules delivered exogenously should optimally be stable within cells until translation of the target RNA has been modulated long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19 (incorporated by reference herein)) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid molecules of the invention results in both enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands. In another embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleic acid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (see for example Wengel et al., International PCT Publication No. WO 00/66604 and WO 99/14226).

In another embodiment, the invention features conjugates and/or complexes of siNA molecules of the invention. Such conjugates and/or complexes can be used to facilitate delivery of siNA molecules into a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acid or non-nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to an siNA molecule of the invention or the sense and antisense strands of an siNA molecule of the invention. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.

The term “biologically active molecule” as used herein, refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active siNA molecules either alone or in combination with other molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof. Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) delivered exogenously optimally are stable within cells until reverse transcription of the RNA has been modulated long enough to reduce the levels of the RNA transcript. The nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and/or in vivo the activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead to better treatments by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules). The treatment of subjects with siNA molecules can also include combinations of different types of nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate, decoys, and aptamers.

In another aspect an siNA molecule of the invention comprises one or more 5′ and/or a 3′-cap structure, for example on only the sense siNA strand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5′-terminus (5′-cap) or at the 3′-terminal (3′-cap) or may be present on both termini. In non-limiting examples, the 5′-cap includes, but is not limited to, glyceryl, inverted deoxy abasic residue (moiety); 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety.

Non-limiting examples of the 3′-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).

By the term “non-nucleotide” is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino, or SH. The term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includes alkynyl groups that have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino or SH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and suitable heterocyclic groups include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylaryl or hydrogen.

“Nucleotide” as used herein, and as recognized in the art, includes natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1′ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents.

In one embodiment, the invention features modified siNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1′ position, see for example Adamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1 carbon of β-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate. Non-limiting examples of modified nucleotides are shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′—NH₂ or 2′-O—NH₂, which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878, which are both incorporated by reference in their entireties.

Various modifications to nucleic acid siNA structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.

Administration of Nucleic Acid Molecules

An siNA molecule of the invention can be adapted for use to prevent or treat cancers and other proliferative conditions, viral infection, inflammatory disease, autoimmunity, respiratory disease, pulmonary disease, cardiovascular disease, neurological disease, renal disease, ocular disease, liver disease, mitochondrial disease, endocrine disease, prion disease, reproduction related diseases and conditions, and/or any other trait, disease or condition that is related to or will respond to the levels of interleukin and/or interleukin receptor in a cell or tissue, alone or in combination with other therapies. For example, an siNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all of which are incorporated herein by reference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and US Patent Application Publication No. US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). In another embodiment, the nucleic acid molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.

In one embodiment, an siNA molecule of the invention is complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No. 20010007666, incorporated by reference herein in its entirety including the drawings. In another embodiment, the membrane disruptive agent or agents and the siNA molecule are also complexed with a cationic lipid or helper lipid molecule, such as those lipids described in U.S. Pat. No. 6,235,310, incorporated by reference herein in its entirety including the drawings.

In one embodiment, an siNA molecule of the invention is complexed with delivery systems as described in U.S. Patent Application Publication No. 2003077829 and International PCT Publication Nos. WO 00/03683 and WO 02/087541, all incorporated by reference herein in their entirety including the drawings.

In addition, the invention features the use of methods to deliver the nucleic acid molecules of the instant invention to the central nervous system and/or peripheral nervous system. Experiments have demonstrated the efficient in vivo uptake of nucleic acids by neurons. As an example of local administration of nucleic acids to nerve cells, Sommer et al., 1998, Antisense Nuc. Acid Drug Dev., 8, 75, describe a study in which a 15mer phosphorothioate antisense nucleic acid molecule to c-fos is administered to rats via microinjection into the brain. Antisense molecules labeled with tetramethylrhodamine-isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by exclusively by neurons thirty minutes post-injection. A diffuse cytoplasmic staining and nuclear staining was observed in these cells. As an example of systemic administration of nucleic acid to nerve cells, Epa et al., 2000, Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo mouse study in which beta-cyclodextrin-adamantane-oligonucleotide conjugates were used to target the p75 neurotrophin receptor in neuronally differentiated PC12 cells. Following a two week course of IP administration, pronounced uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells. In addition, a marked and consistent down-regulation of p75 was observed in DRG neurons. Additional approaches to the targeting of nucleic acid to neurons are described in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle et al., 1997, Eur. J. Pharmocol., 340(2/3), 153; Bannai et al., 1998, Brain Research, 784(1, 2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199; Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, Brain Res. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39. Nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells that express repeat expansion allelic variants for modulation of RE gene expression. The delivery of nucleic acid molecules of the invention, targeting RE is provided by a variety of different strategies. Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier. Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. Furthermore, gene therapy approaches, for example as described in Kaplitt et al., U.S. Pat. No. 6,180,613 and Davidson, WO 04/013280, can be used to express nucleic acid molecules in the CNS.

In addition, the invention features the use of methods to deliver the nucleic acid molecules of the instant invention to hematopoietic cells, including monocytes and lymphocytes. These methods are described in detail by Hartmann et al., 1998, J. Phamacol. Exp. Ther., 285(2), 920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862; Filion and Phillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei, 1996, Leuk. Res., 20(11/12), 925-930; and Bongartz et al., 1994, Nucleic Acids Research, 22(22), 4681-8. Such methods, as described above, include the use of free oligonucleotide, cationic lipid formulations, liposome formulations including pH sensitive liposomes and immunoliposomes, and bioconjugates including oligonucleotides conjugated to fusogenic peptides, for the transfection of hematopoietic cells with oligonucleotides.

In one embodiment, a compound, molecule, or composition for the treatment of ocular conditions (e.g., macular degeneration, diabetic retinopathy etc.) is administered to a subject intraocularly or by intraocular means. In another embodiment, a compound, molecule, or composition for the treatment of ocular conditions (e.g., macular degeneration, diabetic retinopathy etc.) is administered to a subject periocularly or by periocular means (see for example Ahlheim et al., International PCT publication No. WO 03/24420). In one embodiment, an siNA molecule and/or formulation or composition thereof is administered to a subject intraocularly or by intraocular means. In another embodiment, an siNA molecule and/or formulation or composition thereof is administered to a subject periocularly or by periocular means. Periocular administration generally provides a less invasive approach to administering siNA molecules and formulation or composition thereof to a subject (see for example Ahlheim et al., International PCT publication No. WO 03/24420). The use of periocular administration also minimizes the risk of retinal detachment, allows for more frequent dosing or administration, provides a clinically relevant route of administration for macular degeneration and other optic conditions, and also provides the possibility of using reservoirs (e.g., implants, pumps or other devices) for drug delivery.

In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered directly or topically (e.g., locally) to the dermis or follicles as is generally known in the art (see for example Brand, 2001, Curr. Opin. Mol. Ther., 3, 244-8; Regnier et al., 1998, J. Drug Target, 5, 275-89; Kanikkannan, 2002, BioDrugs, 16, 339-47; Wraight et al., 2001, Pharmacol. Ther., 90, 89-104; and Preat and Dujardin, 2001, STP PharmaSciences, 11, 57-68.

In one embodiment, dermal delivery systems of the invention include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. Examples of liposomes which can be used in this invention include the following: (1) CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate) (Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL).

In one embodiment, transmucosal delivery systems of the invention include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).

In one embodiment, nucleic acid molecules of the invention are administered to the central nervous system (CNS) or peripheral nervous system (PNS). Experiments have demonstrated the efficient in vivo uptake of nucleic acids by neurons. As an example of local administration of nucleic acids to nerve cells, Sommer et al., 1998, Antisense Nuc. Acid Drug Dev., 8, 75, describe a study in which a 15mer phosphorothioate antisense nucleic acid molecule to c-fos is administered to rats via microinjection into the brain. Antisense molecules labeled with tetramethylrhodamine-isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by exclusively by neurons thirty minutes post-injection. A diffuse cytoplasmic staining and nuclear staining was observed in these cells. As an example of systemic administration of nucleic acid to nerve cells, Epa et al., 2000, Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo mouse study in which beta-cyclodextrin-adamantane-oligonucleotide conjugates were used to target the p75 neurotrophin receptor in neuronally differentiated PC12 cells. Following a two week course of IP administration, pronounced uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells. In addition, a marked and consistent down-regulation of p75 was observed in DRG neurons. Additional approaches to the targeting of nucleic acid to neurons are described in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle et al., 1997, Eur. J. Pharmocol., 340(2/3), 153; Bannai et al., 1998, Brain Research, 784(1, 2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199; Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, Brain Res. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39. Nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells in the CNS and/or PNS.

The delivery of nucleic acid molecules of the invention to the CNS is provided by a variety of different strategies. Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier. Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. Furthermore, gene therapy approaches, for example as described in Kaplitt et al., U.S. Pat. No. 6,180,613 and Davidson, WO 04/013280, can be used to express nucleic acid molecules in the CNS.

In one embodiment, the nucleic acid molecules of the invention are administered via pulmonary delivery, such as by inhalation of an aerosol or spray dried formulation administered by an inhalation device or nebulizer, providing rapid local uptake of the nucleic acid molecules into relevant pulmonary tissues. Solid particulate compositions containing respirable dry particles of micronized nucleic acid compositions can be prepared by grinding dried or lyophilized nucleic acid compositions, and then passing the micronized composition through, for example, a 400 mesh screen to break up or separate out large agglomerates. A solid particulate composition comprising the nucleic acid compositions of the invention can optionally contain a dispersant which serves to facilitate the formation of an aerosol as well as other therapeutic compounds. A suitable dispersant is lactose, which can be blended with the nucleic acid compound in any suitable ratio, such as a 1 to 1 ratio by weight.

Aerosols of liquid particles comprising a nucleic acid composition of the invention can be produced by any suitable means, such as with a nebulizer (see for example U.S. Pat. No. 4,501,729). Nebulizers are commercially available devices which transform solutions or suspensions of an active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable formulations for use in nebulizers comprise the active ingredient in a liquid carrier in an amount of up to 40% w/w preferably less than 20% w/w of the formulation. The carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride or other suitable salts. Optional additives include preservatives if the formulation is not prepared sterile, for example, methyl hydroxybenzoate, anti-oxidants, flavorings, volatile oils, buffering agents and emulsifiers and other formulation surfactants. The aerosols of solid particles comprising the active composition and surfactant can likewise be produced with any solid particulate aerosol generator. Aerosol generators for administering solid particulate therapeutics to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a therapeutic composition at a rate suitable for human administration. One illustrative type of solid particulate aerosol generator is an insufflator. Suitable formulations for administration by insufflation include finely comminuted powders which can be delivered by means of an insufflator. In the insufflator, the powder, e.g., a metered dose thereof effective to carry out the treatments described herein, is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically comprises from 0.1 to 100 w/w of the formulation. A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquefied propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation can additionally contain one or more co-solvents, for example, ethanol, emulsifiers and other formulation surfactants, such as oleic acid or sorbitan trioleate, anti-oxidants and suitable flavoring agents. Other methods for pulmonary delivery are described in, for example US Patent Application No. 20040037780, and U.S. Pat. Nos. 6,592,904; 6,582,728; 6,565,885.

In one embodiment, siNA molecules of the invention are formulated or complexed with polyethylenimine (e.g., linear or branched PEI) and/or polyethylenimine derivatives, including for example grafted PEIs such as galactose PEI, cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI (PEG-PEI) derivatives thereof (see for example Ogris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003, Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, Pharmaceutical Research, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22, 46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Peterson et al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999, Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNAS USA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release, 60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274, 19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; and Sagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, an siNA molecule of the invention comprises a bioconjugate, for example a nucleic acid conjugate as described in Vargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S. Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat. No. 6,235,886; U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S. Pat. No. 5,138,045, all incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising one or more nucleic acid(s) of the invention in an acceptable carrier, such as a stabilizer, buffer, and the like. The polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration, and the other compositions known in the art.

The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes that lead to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the siNA molecules of the invention to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.

By “pharmaceutically acceptable formulation” is meant a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85), which can enhance entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge, Mass.); and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985), hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.

Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.

It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable for administering nucleic acid molecules of the invention to specific cell types. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). In another example, the folate receptor is overexpressed in many cancer cells. Binding of such glycoproteins, synthetic glycoconjugates, or folates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatennary or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose, galactosamine, or folate based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to, for example, the treatment of liver disease, cancers of the liver, or other cancers. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention. Non-limiting examples of such bioconjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser. No. 10/151,116, filed May 17, 2002. In one embodiment, nucleic acid molecules of the invention are complexed with or covalently attached to nanoparticles, such as Hepatitis B virus S, M, or L evelope proteins (see for example Yamado et al., 2003, Nature Biotechnology, 21, 885). In one embodiment, nucleic acid molecules of the invention are delivered with specificity for human tumor cells, specifically non-apoptotic human tumor cells including for example T-cells, hepatocytes, breast carcinoma cells, ovarian carcinoma cells, melanoma cells, intestinal epithelial cells, prostate cells, testicular cells, non-small cell lung cancers, small cell lung cancers, etc.

In one embodiment, an siNA molecule of the invention is designed or formulated to specifically target endothelial cells or tumor cells. For example, various formulations and conjugates can be utilized to specifically target endothelial cells or tumor cells, including PEI-PEG-folate, PEI-PEG-RGD, PEI-PEG-biotin, PEI-PEG-cholesterol, and other conjugates known in the art that enable specific targeting to endothelial cells and/or tumor cells.

Alternatively, certain siNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; propulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45). Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856).

In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, pol III based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pats. Nos. 5,902,880 and 6,146,886). The recombinant vectors capable of expressing the siNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).

In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the instant invention. The expression vector can encode one or both strands of an siNA duplex, or a single self-complementary strand that self hybridizes into an siNA duplex. The nucleic acid sequences encoding the siNA molecules of the instant invention can be operably linked in a manner that allows expression of the siNA molecule (see for example Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi: 10.1038/nm725).

In another aspect, the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the instant invention, wherein said sequence is operably linked to said initiation region and said termination region in a manner that allows expression and/or delivery of the siNA molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the siNA of the invention; and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S. A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siNA in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736. The above siNA transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the siNA molecules of the invention in a manner that allows expression of that siNA molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; and c) a nucleic acid sequence encoding at least one strand of the siNA molecule, wherein the sequence is operably linked to the initiation region and the termination region in a manner that allows expression and/or delivery of the siNA molecule.

In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; and d) a nucleic acid sequence encoding at least one strand of an siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the open reading frame and the termination region in a manner that allows expression and/or delivery of the siNA molecule. In yet another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; and d) a nucleic acid sequence encoding at least one siNA molecule, wherein the sequence is operably linked to the initiation region, the intron and the termination region in a manner which allows expression and/or delivery of the nucleic acid molecule.

In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; and e) a nucleic acid sequence encoding at least one strand of an siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the intron, the open reading frame and the termination region in a manner which allows expression and/or delivery of the siNA molecule.

Interleukin Biology and Biochemistry

The following discussion is adapted from R&D Systems Mini-Reviews and Tech Notes, Cytokine Mini-Reviews, Copyright©2002 R&D Systems. Interleukin 2 (IL-2) is a lymphokine synthesized and secreted primarily by T helper lymphocytes that have been activated by stimulation with certain mitogens or by interaction of the T cell receptor complex with antigen/MHC complexes on the surfaces of antigen-presenting cells. The response of T helper cells to activation is induction of the expression of IL-2 and receptors for IL-2 and, subsequently, clonal expansion of antigen-specific T cells. At this level IL-2 is an autocrine factor, driving the expansion of the antigen-specific cells. IL-2 also acts as a paracrine factor, influencing the activity of other cells, both within the immune system and outside of it. B cells and natural killer (NK) cells respond, when properly activated, to IL-2. The so-called lymphocyte activated killer, or LAK cells, appear to be derived from NK cells under the influence of IL-2.

The biological activities of IL-2 are mediated through the binding of IL-2 to a multisubunit cellular receptor. Although three distinct transmembrane glycoprotein subunits contribute to the formation of the high affinity IL-2 receptor, various combinations of receptor subunits (alpha, beta, gamma) are known to occur.

Interleukin 1 (IL-1) is a general name for two distinct proteins, IL-1a and IL-1b, that are considered the first of a family of regulatory and inflammatory cytokines. Along with IL-1 receptor antagonist (IL-1ra)₂ and IL-18,3 these molecules play important roles in the up- and down-regulation of acute inflammation. In the immune system, the production of IL-1 is typically induced, generally resulting in inflammation. IL-1b and TNF-a are generally thought of as prototypical pro-inflammatory cytokines. The effects of IL-1, however, are not limited to inflammation, as IL-1 has also been associated with bone formation and remodeling, insulin secretion, appetite regulation, fever induction, neuronal phenotype development, and IGF/GH physiology. IL-1 has also been known by a number of alternative names, including lymphocyte activating factor, endogenous pyrogen, catabolin, hemopoietin-1, melanoma growth inhibition factor, and osteoclast activating factor. IL-1a and IL-1b exert their effects by binding to specific receptors. Two distinct IL-1 receptor binding proteins, plus a non-binding signaling accessory protein have been identified to date. Each have three extracellular immunoglobulin-like (Ig-like) domains, qualifying them for membership in the type IV cytokine receptor family.

Interleukin-4 (IL-4) mediates important pro-inflammatory functions in asthma including induction of the IgE isotype switch, expression of vascular cell adhesion molecule-1 (VCAM-1), promotion of eosinophil transmigration across endothelium, mucus secretion, and differentiation of T helper type 2 lymphocytes leading to cytokine release. Asthma has been linked to polymorphisms in the IL-4 gene promoter and proteins involved in IL-4 signaling. Soluble recombinant IL-4 receptor lacks transmembrane and cytoplasmic activating domains and can therefore sequester IL-4 without mediating cellular activation. Genetic variants within the IL-4 signaling pathway might contribute to the risk of developing asthma in a given individual. A number of polymorphisms have been described within the IL-4 receptor a (IL-4Rα) gene, and in addition, polymorphism occurs in the promoter for the IL-4 gene itself (see for example Hall, 2000, Respir. Res., 1, 6-8 and Ober et al., 2000, Am J Hum Genet., 66, 517-526, for a review). The type 2 cytokine IL-13, which shares a receptor component and signaling pathways with IL-4, was found to be necessary and sufficient for the expression of allergic asthma (see Wills-Karp et al., 1998, Science, 282, 2258-61). IL-13 induces the pathophysiological features of asthma in a manner that is independent of immunoglobulin E and eosinophils. Thus, IL-13 is critical to allergen-induced asthma but operates through mechanisms other than those that are classically implicated in allergic responses.

Human IL-5 is a 134 amino acid polypeptide with a predicted mass of 12.5 kDa. It is secreted by a restricted number of mesenchymal cell types. In its native state, mature IL-5 is synthesized as a 115 aa, highly glycosylated 22 kDa monomer that forms a 40-50 kDa disulfide-linked homodimer. Although the content of carbohydrate is high, carbohydrate is not needed for bioactivity. Monomeric IL-5 has no activity; a homodimer is required for function. This is in contrast to the receptor-related cytokines IL-3 and GM-CSF, which exist only as monomers. Just as one IL-3 and GM-CSF monomer binds to one receptor, one IL-5 homodimer is able to engage only one IL-5 receptor. It has been suggested that IL-5 (as a dimer) undergoes a general conformational change after binding to one receptor molecule, and this change precludes binding to a second receptor. The receptor for IL-5 consists of a ligand binding a-subunit and a non-ligand binding (common) signal transducing b-subunit that is shared by the receptors for IL-3 and GM-CSF. IL-5 appears to perform a number of functions on eosinophils. These include the down modulation of Mac-1, the upregulation of receptors for IgA and IgG, the stimulation of lipid mediator (leukotriene C4 and PAF) secretion and the induction of granule release. IL-5 also promotes the growth and differentiation of eosinophils.

Interleukin 6 (IL-6) is considered a prototypic pleiotrophic cytokine. This is reflected in the variety of names originally assigned to IL-6 based on function, including Interferon b2, IL-1-inducible 26 kD Protein, Hepatocyte Stimulating Factor, Cytotoxic T-cell Differentiation Factor, B cell Differentiation Factor (BCDF) and/or B cell Stimulatory Factor 2 (BSF2). A number of cytokines make up an IL-6 cytokine family. Membership in this family is typically based on a helical cytokine structure and receptor subunit makeup. The functional receptor for IL-6 is a complex of two transmembrane glycoproteins (gp130 and IL-6 receptor) that are members of the Class I cytokine receptor superfamily.

Because of the central role of the interleukin family of cytokines in the mediation of immune and inflammatory responses, modulation of interleukin expression and/or activity can provide important functions in therapeutic and diagnostic applications. The use of small interfering nucleic acid molecules targeting interleukins and their corresponding receptors therefore provides a class of novel therapeutic agents that can be used in the treatment of cancers, proliferative diseases, inflammatory disease, respiratory disease, pulmonary disease, cardiovascular disease, autoimmune disease, infectious disease, prior disease, renal disease, transplant rejection, or any other disease or condition that responds to modulation of interleukin and interleukin receptor genes.

EXAMPLES

The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.

Example 1 Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandem using a cleavable linker, for example, a succinyl-based linker. Tandem synthesis as described herein is followed by a one-step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.

After completing a tandem synthesis of an siNA oligo and its complement in which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact (trityl on synthesis), the oligonucleotides are deprotected as described above. Following deprotection, the siNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5′-O-DMT group while the complementary strand comprises a terminal 5′-hydroxyl. The newly formed duplex behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxytrityl group. Because the strands form a stable duplex, this dimethoxytrityl group (or an equivalent group, such as other trityl groups or other hydrophobic moieties) is all that is required to purify the pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point of introducing a tandem linker, such as an inverted deoxy abasic succinate or glyceryl succinate linker (see FIG. 1) or an equivalent cleavable linker. A non-limiting example of linker coupling conditions that can be used includes a hindered base such as diisopropylethylamine (DIPA) and/or DMAP in the presence of an activator reagent such as Bromotripyrrolidinophosphoniumhexafluororophosphate (PyBrOP). After the linker is coupled, standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5′-O-DMT intact. Following synthesis, the resulting oligonucleotide is deprotected according to the procedures described herein and quenched with a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solid phase extraction, for example using a Waters C18 SepPak 1 g cartridge conditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2 CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50 mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with 50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with 1 CV H2O followed by on-column detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CV of 1% aqueous TFA to the column and allowing to stand for approximately 10 minutes. The remaining TFA solution is removed and the column washed with H20 followed by 1 CV 1M NaCl and additional H2O. The siNA duplex product is then eluted, for example, using 1 CV 20% aqueous CAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of a purified siNA construct in which each peak corresponds to the calculated mass of an individual siNA strand of the siNA duplex. The same purified siNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably corresponding to the duplex siNA, and two peaks presumably corresponding to the separate siNA sequence strands. Ion exchange HPLC analysis of the same siNA contract only shows a single peak. Testing of the purified siNA construct using a luciferase reporter assay described below demonstrated the same RNAi activity compared to siNA constructs generated from separately synthesized oligonucleotide sequence strands.

Example 2 Identification of Potential siNA Target Sites in any RNA Sequence

The sequence of an RNA target of interest, such as a viral or human mRNA transcript, is screened for target sites, for example by using a computer folding algorithm. In a non-limiting example, the sequence of a gene or RNA gene transcript derived from a database, such as Genbank, is used to generate siNA targets having complementarity to the target. Such sequences can be obtained from a database, or can be determined experimentally as known in the art. Target sites that are known, for example, those target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease or condition such as those sites containing mutations or deletions, can be used to design siNA molecules targeting those sites. Various parameters can be used to determine which sites are the most suitable target sites within the target RNA sequence. These parameters include but are not limited to secondary or tertiary RNA structure, the nucleotide base composition of the target sequence, the degree of homology between various regions of the target sequence, or the relative position of the target sequence within the RNA transcript. Based on these determinations, any number of target sites within the RNA transcript can be chosen to screen siNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. In a non-limiting example, anywhere from 1 to 1000 target sites are chosen within the transcript based on the size of the siNA construct to be used. High throughput screening assays can be developed for screening siNA molecules using methods known in the art, such as with multi-well or multi-plate assays to determine efficient reduction in target gene expression.

Example 3 Selection of siNA Molecule Target Fites in a RNA

The following non-limiting steps can be used to carry out the selection of siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target sequence. This step is typically carried out using a custom Perl script, but commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be employed as well.

2. In some instances the siNAs correspond to more than one target sequence; such would be the case for example in targeting different transcripts of the same gene, targeting different transcripts of more than one gene, or for targeting both the human gene and an animal homolog. In this case, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find matching sequences in each list. The subsequences are then ranked according to the number of target sequences that contain the given subsequence; the goal is to find subsequences that are present in most or all of the target sequences. Alternately, the ranking can identify subsequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siNA to target specifically the mutant sequence and not effect the expression of the normal sequence.

3. In some instances the siNA subsequences are absent in one or more sequences while present in the desired target sequence; such would be the case if the siNA targets a gene with a paralogous family member that is to remain untargeted. As in case 2 above, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find sequences that are present in the target gene but are absent in the untargeted paralog.

4. The ranked siNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC.

5. The ranked siNA subsequences can be further analyzed and ranked according to self-folding and internal hairpins. Weaker internal folds are preferred; strong hairpin structures are to be avoided.

6. The ranked siNA subsequences can be further analyzed and ranked according to whether they have runs of GGG or CCC in the sequence. GGG (or even more Gs) in either strand can make oligonucleotide synthesis problematic and can potentially interfere with RNAi activity, so it is avoided whenever better sequences are available. CCC is searched in the target strand because that will place GGG in the antisense strand.

7. The ranked siNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide UU (uridine dinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end of the sequence (to yield 3′ UU on the antisense sequence). These sequences allow one to design siNA molecules with terminal TT thymidine dinucleotides.

8. Four or five target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) strand of the siNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) strand of the siNA duplex (see Tables II and III). If terminal TT residues are desired for the sequence (as described in paragraph 7), then the two 3′ terminal nucleotides of both the sense and antisense strands are replaced by TT prior to synthesizing the oligos.

9. The siNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siNA molecule or the most preferred target site within the target RNA sequence.

10. Other design considerations can be used when selecting target nucleic acid sequences, see for example Reynolds et al., 2004, Nature Biotechnology Advanced Online Publication, 1 Feb. 2004, doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32, doi:10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to a interleukin and/or interleukin receptor target sequence is used to screen for target sites in cells expressing interleukin and/or interleukin receptor RNA, such as cultured Jurkat, HeLa, or 293T cells. The general strategy used in this approach is shown in FIG. 9. A non-limiting example of such is a pool comprising sequences having any of SEQ ID NOS1-1828. Cells expressing interleukin and/or interleukin receptor (e.g., Jurkat, HeLa, or 293T cells) are transfected with the pool of siNA constructs and cells that demonstrate a phenotype associated with interleukin and/or interleukin receptor inhibition are sorted. The pool of siNA constructs can be expressed from transcription cassettes inserted into appropriate vectors (see for example FIG. 7 and FIG. 8). The siNA from cells demonstrating a positive phenotypic change (e.g., decreased interleukin and/or interleukin receptor mRNA levels or decreased interleukin and/or interleukin receptor protein expression), are sequenced to determine the most suitable target site(s) within the target interleukin and/or interleukin receptor RNA sequence.

Example 4 Interleukin and Interleukin Receptor Targeted siNA Design

siNA target sites were chosen by analyzing sequences of the interleukin and/or interleukin receptor RNA target and optionally prioritizing the target sites on the basis of folding (structure of any given sequence analyzed to determine siNA accessibility to the target), by using a library of siNA molecules as described in Example 3, or alternately by using an in vitro siNA system as described in Example 6 herein. siNA molecules were designed that could bind each target and are optionally individually analyzed by computer folding to assess whether the siNA molecule can interact with the target sequence. Varying the length of the siNA molecules can be chosen to optimize activity. Generally, a sufficient number of complementary nucleotide bases are chosen to bind to, or otherwise interact with, the target RNA, but the degree of complementarity can be modulated to accommodate siNA duplexes or varying length or base composition. By using such methodologies, siNA molecules can be designed to target sites within any known RNA sequence, for example those RNA sequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nuclease stability for systemic administration in vivo and/or improved pharmacokinetic, localization, and delivery properties while preserving the ability to mediate RNAi activity. Chemical modifications as described herein are introduced synthetically using synthetic methods described herein and those generally known in the art. The synthetic siNA constructs are then assayed for nuclease stability in serum and/or cellular/tissue extracts (e.g. liver extracts). The synthetic siNA constructs are also tested in parallel for RNAi activity using an appropriate assay, such as a luciferase reporter assay as described herein or another suitable assay that can quantity RNAi activity. Synthetic siNA constructs that possess both nuclease stability and RNAi activity can be further modified and re-evaluated in stability and activity assays. The chemical modifications of the stabilized active siNA constructs can then be applied to any siNA sequence targeting any chosen RNA and used, for example, in target screening assays to pick lead siNA compounds for therapeutic development (see for example FIG. 11).

Example 5 Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein. The sequence of one strand of the siNA molecule(s) is complementary to the target site sequences described above. The siNA molecules can be chemically synthesized using methods described herein. Inactive siNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the siNA molecules such that it is not complementary to the target sequence. Generally, siNA constructs can by synthesized using solid phase oligonucleotide synthesis methods as described herein (see for example Usman et al., U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein in their entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in a stepwise fashion using the phosphoramidite chemistry as is known in the art. Standard phosphoramidite chemistry involves the use of nucleosides comprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl, 3′-O-2-Cyanoethyl N,N-diisopropylphos-phoroamidite groups, and exocyclic amine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be used in conjunction with acid-labile 2′-O-orthoester protecting groups in the synthesis of RNA as described by Scaringe supra. Differing 2′ chemistries can require different protecting groups, for example 2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection as described by Usman et al., U.S. Pat. No. 5,631,360, incorporated by reference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′- to 5′-direction) to the solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support (e.g., controlled pore glass or polystyrene) using various linkers. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are combined resulting in the coupling of the second nucleoside phosphoramidite onto the 5′-end of the first nucleoside. The support is then washed and any unreacted 5′-hydroxyl groups are capped with a capping reagent such as acetic anhydride to yield inactive 5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized to a more stable phosphate linkage. At the end of the nucleotide addition cycle, the 5′-O-protecting group is cleaved under suitable conditions (e.g., acidic conditions for trityl-based groups and Fluoride for silyl-based groups). The cycle is repeated for each subsequent nucleotide.

Modification of synthesis conditions can be used to optimize coupling efficiency, for example by using differing coupling times, differing reagent/phosphoramidite concentrations, differing contact times, differing solid supports and solid support linker chemistries depending on the particular chemical composition of the siNA to be synthesized. Deprotection and purification of the siNA can be performed as is generally described in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat. No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No. 6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or Scaringe supra, incorporated by reference herein in their entireties. Additionally, deprotection conditions can be modified to provide the best possible yield and purity of siNA constructs. For example, applicant has observed that oligonucleotides comprising 2′-deoxy-2′-fluoro nucleotides can degrade under inappropriate deprotection conditions. Such oligonucleotides are deprotected using aqueous methylamine at about 35° C. for 30 minutes. If the 2′-deoxy-2′-fluoro containing oligonucleotide also comprises ribonucleotides, after deprotection with aqueous methylamine at about 35° C. for 30 minutes, TEA-HF is added and the reaction maintained at about 65° C. for an additional 15 minutes.

Example 6 RNAi In Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is used to evaluate siNA constructs targeting interleukin and/or interleukin receptor RNA targets. The assay comprises the system described by Tuschl et al., 1999, Genes and Development, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use with interleukin and/or interleukin receptor target RNA. A Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro. Target RNA is generated via in vitro transcription from an appropriate interleukin and/or interleukin receptor expressing plasmid using T7 RNA polymerase or via chemical synthesis as described herein. Sense and antisense siNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide. The Drosophila lysate is prepared using zero to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10% [vol/vol] lysis buffer containing siNA (10 nM final concentration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug/ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 25° C. for 10 minutes before adding RNA, then incubated at 25° C. for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25× Passive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which siNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [alpha-³²P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5′-³²P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by PHOSPHOR IMAGER® (autoradiography) quantitation of bands representing intact control RNA or RNA from control reactions without siNA and the cleavage products generated by the assay.

In one embodiment, this assay is used to determine target sites the interleukin and/or interleukin receptor RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA constructs are screened for RNAi mediated cleavage of the interleukin and/or interleukin receptor RNA target, for example, by analyzing the assay reaction by electrophoresis of labeled target RNA, or by Northern blotting, as well as by other methodology well known in the art.

Example 7 Nucleic Acid Inhibition of Interleukin and Interleukin Receptor Target RNA in Vitro

siNA molecules targeted to the human interleukin and/or interleukin receptor RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. The target sequences and the nucleotide location within the interleukin and/or interleukin receptor RNA are given in Table II and III.

Two formats are used to test the efficacy of siNAs targeting interleukin and/or interleukin receptor. First, the reagents are tested in cell culture using, for example, Jurkat, HeLa, or 293T cells, to determine the extent of RNA and protein inhibition. siNA reagents (e.g.; see Tables II and III) are selected against the interleukin and/or interleukin receptor target as described herein. RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to, for example, cultured Jurkat, HeLa, or 293T cells. Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification (e.g., ABI 7700 TAQMAN®). A comparison is made to a mixture of oligonucleotide sequences made to unrelated targets or to a randomized siNA control with the same overall length and chemistry, but randomly substituted at each position. Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent concentration is chosen, a RNA time-course of inhibition is performed with the lead siNA molecule. In addition, a cell-plating format can be used to determine RNA inhibition.

Delivery of siNA to Cells

Cells (e.g., Jurkat, HeLa, or 293T cells) are seeded, for example, at 1×10⁵ cells per well of a six-well dish in EGM-2 (BioWhittaker) the day before transfection. siNA (final concentration, for example 20 nM) and cationic lipid (e.g., final concentration 2 μg/ml) are complexed in EGM basal media (Bio Whittaker) at 37° C. for 30 minutes in polystyrene tubes. Following vortexing, the complexed siNA is added to each well and incubated for the times indicated. For initial optimization experiments, cells are seeded, for example, at 1×10³ in 96 well plates and siNA complex added as described. Efficiency of delivery of siNA to cells is determined using a fluorescent siNA complexed with lipid. Cells in 6-well dishes are incubated with siNA for 24 hours, rinsed with PBS and fixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptake of siNA is visualized using a fluorescent microscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and Lightcycler Quantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example, using Qiagen RNA purification kits for 6-well or Rneasy extraction kits for 96-well assays. For TAQMAN® analysis (real-time PCR monitoring of amplification), dual-labeled probes are synthesized with the reporter dye, FAM or JOE, covalently linked at the 5′-end and the quencher dye TAMRA conjugated to the 3′-end. One-step RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence Detector using 50 μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900 nM reverse primer, 100 nM probe, 1× TaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM each dATP, dCTP, dGTP, and dTTP, 10U RNase Inhibitor (Promega), 1.25U AMPLITAQ GOLD® (DNA polymerase) (PE-Applied Biosystems) and 10U M-MLV Reverse Transcriptase (Promega). The thermal cycling conditions can consist of 30 minutes at 48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. Quantitation of mRNA levels is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 33, 11 ng/r×n) and normalizing to β-actin or GAPDH mRNA in parallel TAQMAN® reactions (real-time PCR monitoring of amplification). For each gene of interest an upper and lower primer and a fluorescently labeled probe are designed. Real time incorporation of SYBR Green I dye into a specific PCR product can be measured in glass capillary tubes using a lightcyler. A standard curve is generated for each primer pair using control cRNA. Values are represented as relative expression to GAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparation technique (see for example Andrews and Faller, 1991, Nucleic Acids Research, 19, 2499). Protein extracts from supernatants are prepared, for example using TCA precipitation. An equal volume of 20% TCA is added to the cell supernatant, incubated on ice for 1 hour and pelleted by centrifugation for 5 minutes. Pellets are washed in acetone, dried and resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts) polyacrylamide gel and transferred onto nitro-cellulose membranes. Non-specific binding can be blocked by incubation, for example, with 5% non-fat milk for 1 hour followed by primary antibody for 16 hour at 4° C. Following washes, the secondary antibody is applied, for example (1:10,000 dilution) for 1 hour at room temperature and the signal detected with SuperSignal reagent (Pierce).

Example 8 Animal Models Useful to Evaluate the Down-Regulation of Interleukin and/or Interleukin Receptor Gene Expression

Evaluating the efficacy of anti-interleukin agents in animal models is an important prerequisite to human clinical trials. Allogeneic rejection is the most common cause of corneal graft failure. King et al., 2000, Transplantation, 70, 1225-1233, describe a study investigating the kinetics of cytokine and chemokine mRNA expression before and after the onset of corneal graft rejection. Intracorneal cytokine and chemokine mRNA levels were investigated in the Brown Norway-Lewis inbred rat model, in which rejection onset is observed at 8/9 days after grafting in all animals. Nongrafted corneas and syngeneic (Lewis-Lewis) corneal transplants were used as controls. Donor and recipient cornea were examined by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) for hypoxanthine phosphoribosyltransferase (HPRT), CD3, CD25, interleukin (IL)-1beta, IL-IRA, IL-2, IL-6, IL-10, interferon-gamma (IFN-gamma), tumor necrosis factor (TNF), transforming growth factor (TGF)-beta1, and macrophage inflammatory protein (MIP)-2 and by RT-PCR for IL-4, IL-5, IL-12 p40, IL-13, TGF-beta.2, monocyte chemotactic protein-1 (MCP-1), MIP-1alpha, MIP-1beta, and RANTES. A biphasic expression of cytokine and chemokine mRNA was found after transplantation. During the early phase (days 3-9), there was an elevation of the majority of the cytokines examined, including IL-1beta, IL-6, IL-10, IL-12 p40, and MIP-2. There was no difference in cytokine expression patterns between allogeneic or syngeneic recipients at this time. In syngeneic recipients, cytokine levels reduced to pretransplant levels by day 13, whereas levels of all cytokines rose after the rejection onset in the allografts, including TGF-beta.1, TGF-beta.2, and IL-IRA. The T cell-derived cytokines IL-4, IL-13, and IFN-gamma were detected only during the rejection phase in allogeneic recipients. Thus, there appears to be an early cytokine and chemokine response to the transplantation process, evident in syngeneic and allogeneic grafts, that drives angiogenesis, leukocyte recruitment, and affects other leukocyte functions. After an immune response has been generated, allogeneic rejection results in the expression of Th1 cytokines, Th2 cytokines, and anti-inflammatory/Th3 cytokines. This animal model can be used to evaluate the efficacy of nucleic acid molecules of the invention targeting interleukin expression (e.g., phenotypic change, interleukin expression etc.) toward therapeutic use in treating transplant rejection. Similarly, other animal models of transplant rejection as are known in the art can be used to evaluate nucleic acid molecules (e.g., siNA) of the invention toward therapeutic use.

Other animal models are useful in evaluating the role of interleukins in asthma. For example, Kuperman et al., 2002, Nature Medicine, 8, 885-9, describe an animal model of IL-13 mediated asthma response animal models of allergic asthma in which blockade of IL-13 markedly inhibits allergen-induced asthma. Venkayya et al., 2002, Am J Respir Cell Mol Biol., 26, 202-8 and Yang et al., 2001, Am J Respir Cell Mol Biol., 25, 522-30 describe animal models of airway inflammation and airway hyperresponsiveness (AHR) in which IL-4/IL-4R and IL-13 mediate asthma. These models can be used to evaluate the efficacy of siNA molecules of the invention targeting, for example, IL-4, IL-4R, IL-13, and/or IL-13R for use is treating asthma.

Example 9 RNAi Mediated Inhibition of Interleukin and/or Interleukin Receptor Expression in Cell Culture

Inhibition of Interleukin and/or Interleukin Receptor RNA Expression Using siNA Targeting Interleukin and/or Interleukin Receptor RNA

siNA constructs (Table III) are tested for efficacy in reducing interleukin and/or interleukin receptor RNA expression in, for example, Jurkat, HeLa, or 293T cells. Cells are plated approximately 24 hours before transfection in 96-well plates at 5,000-7,500 cells/well, 100 μl/well, such that at the time of transfection cells are 70-90% confluent. For transfection, annealed siNAs are mixed with the transfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 0.5 μl/well and incubated for 20 min. at room temperature. The siNA transfection mixtures are added to cells to give a final siNA concentration of 25 nM in a volume of 150 μl. Each siNA transfection mixture is added to 3 wells for triplicate siNA treatments. Cells are incubated at 37° for 24 h in the continued presence of the siNA transfection mixture. At 24 h, RNA is prepared from each well of treated cells. The supernatants with the transfection mixtures are first removed and discarded, then the cells are lysed and RNA prepared from each well. Target gene expression following treatment is evaluated by RT-PCR for the target gene and for a control gene (36B4, an RNA polymerase subunit) for normalization. The triplicate data is averaged and the standard deviations determined for each treatment. Normalized data are graphed and the percent reduction of target mRNA by active siNAs in comparison to their respective inverted control siNAs is determined.

In a non-limiting example, chemically modified siNA constructs (Table III) were tested for efficacy as described above in reducing IL-4R RNA expression in HeLa cells. Active siNAs were evaluated compared to a matched chemistry inverted control (IC), and a transfection control. Results are summarized in FIG. 22. FIG. 22 shows results for Stab 9/22 (Table IV) siNA constructs targeting various sites in IL-4R mRNA. As shown in FIG. 22, the active siNA constructs provide significant inhibition of IL-4R gene expression in cell culture experiments as determined by levels of IL-4R mRNA when compared to appropriate controls.

Example 10 Indications

The siNA molecule of the invention can be used to prevent, inhibit or treat cancers and other proliferative conditions, viral infection, inflammatory disease, autoimmunity, respiratory disease, pulmonary disease, cardiovascular disease, neurological disease, renal disease, ocular disease, liver disease, mitochondrial disease, endocrine disease, prion disease, reproduction related diseases and conditions, and/or any other trait, disease or condition that is related to or will respond to the levels of interleukin and/or interleukin receptor in a cell or tissue, alone or in combination with other therapies. Non-limiting examples of respiratory diseases that can be treated using siNA molecules of the invention (e.g., siNA molecules targeting IL-4, IL-4R, IL-13, and/or IL-13R include asthma, chronic obstructive pulmonary disease or “COPD”, allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation, allergies, impeded respiration, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema.

The use of anticholinergic agents, anti-inflammatories, bronchodilators, adenosine inhibitors, adenosine A1 receptor inhibitors, non-selective M3 receptor antagonists such as atropine, ipratropium bromide and selective M3 receptor antagonists such as darifenacin and revatropate are all non-limiting examples of agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA molecules) of the instant invention. Immunomodulators, chemotherapeutics, anti-inflammatory compounds, and anti-viral compounds are additional non-limiting examples of pharmaceutical agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA molecules) of the instant invention. Those skilled in the art will recognize that other drugs, compounds and therapies can similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. siRNA molecules) are hence within the scope of the instant invention.

Example 11 Diagnostic Uses

The siNA molecules of the invention can be used in a variety of diagnostic applications, such as in the identification of molecular targets (e.g., RNA) in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings. Such diagnostic use of siNA molecules involves utilizing reconstituted RNAi systems, for example, using cellular lysates or partially purified cellular lysates. siNA molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell. The close relationship between siNA activity and the structure of the target RNA allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple siNA molecules described in this invention, one can map nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with siNA molecules can be used to inhibit gene expression and define the role of specified gene products in the progression of disease or infection. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes, siNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations siNA molecules and/or other chemical or biological molecules). Other in vitro uses of siNA molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with an siNA using standard methodologies, for example, fluorescence resonance emission transfer (FRET).

In a specific example, siNA molecules that cleave only wild-type or mutant forms of the target RNA are used for the assay. The first siNA molecules (i.e., those that cleave only wild-type forms of target RNA) are used to identify wild-type RNA present in the sample and the second siNA molecules (i.e., those that cleave only mutant forms of target RNA) are used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both siNA molecules to demonstrate the relative siNA efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus, each analysis requires two siNA molecules, two substrates and one unknown sample, which is combined into six reactions. The presence of cleavage products is determined using an RNase protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., disease related or infection related) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels is adequate and decreases the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The present invention teaches one skilled in the art to test various combinations and/or substitutions of chemical modifications described herein toward generating nucleic acid constructs with improved activity for mediating RNAi activity. Such improved activity can comprise improved stability, improved bioavailability, and/or improved activation of cellular responses mediating RNAi. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying siNA molecules with improved RNAi activity.

The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

TABLE I interleukin and/or interleukin receptor Accession Numbers Interleukin Family NM_000575 Homo sapiens interleukin 1, alpha (IL1A), mRNA NM_000576 Homo sapiens interleukin 1, beta (IL1B), mRNA NM_012275 Homo sapiens interleukin 1 family, member 5 (delta) (IL1F5), mRNA NM_014440 Homo sapiens interleukin 1 family, member 6 (epsilon) (IL1F6), mRNA NM_014439 Homo sapiens interleukin 1 family, member 7 (zeta) (IL1F7), mRNA NM_014438 Homo sapiens interleukin 1 family, member 8 (eta) (IL1F8), mRNA NM_019618 Homo sapiens interleukin 1 family, member 9 (IL1F9), mRNA NM_032556 Homo sapiens interleukin 1 family, member 10 (theta) (IL1F10), mRNA NM_000586 Homo sapiens interleukin 2 (IL2), mRNA NM_000588 Homo sapiens interleukin 3 (colony-stimulating factor, multiple) (IL3), mRNA NM_000589 Homo sapiens interleukin 4 (IL4), mRNA NM_000879 Homo sapiens interleukin 5 (colony-stimulating factor, eosinophil) (IL5), mRNA NM_000600 Homo sapiens interleukin 6 (interferon, beta 2) (IL6), mRNA NM_000880 Homo sapiens interleukin 7 (IL7), mRNA NM_000584 Homo sapiens interleukin 8 (IL8), mRNA NM_000590 Homo sapiens interleukin 9 (IL9), mRNA NM_000572 Homo sapiens interleukin 10 (IL10), mRNA NM_000641 Homo sapiens interleukin 11 (IL11), mRNA NM_000882 Homo sapiens interleukin 12A (natural killer cell stimulatory factor 1, cytotoxic lymphocyte maturation factor 1, p35) (IL12A), mRNA NM_002187 Homo sapiens interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p40) (IL12B), mRNA NM_002188 Homo sapiens interleukin 13 (IL13), mRNA L15344 Homo sapiens interleukin 14 (IL14), mRNA NM_000585 Homo sapiens interleukin 15 (IL15), mRNA NM_004513 Homo sapiens interleukin 16 (lymphocyte chemoattractant factor) (IL16), mRNA NM_002190 Homo sapiens interleukin 17 (cytotoxic T-lymphocyte-associated serine esterase 8) (IL17), mRNA NM_014443 Homo sapiens interleukin 17B (IL17B), mRNA NM_013278 Homo sapiens interleukin 17C (IL17C), mRNA NM_138284 Homo sapiens interleukin 17D (IL17D), mRNA NM_022789 Homo sapiens interleukin 17E (IL17E), mRNA NM_052872 Homo sapiens interleukin 17F (IL17F), mRNA NM_001562 Homo sapiens interleukin 18 (interferon-gamma-inducing factor) (IL18), mRNA NM_013371 Homo sapiens interleukin 19 (IL19), mRNA NM_018724 Homo sapiens interleukin 20 (IL20), mRNA NM_021803 Homo sapiens interleukin 21 (IL21 antisense), mRNA NM_020525 Homo sapiens interleukin 22 (IL22), mRNA NM_016584 Homo sapiens interleukin 23, alpha subunit p19 (IL23A), mRNA NM_006850 Homo sapiens interleukin 24 (IL24), mRNA NM_018402 Homo sapiens interleukin 26 (IL26), mRNA AL365373 Homo sapiens interleukin 27 (IL27), mRNA Interleukin Receptor Family NM_000877 Homo sapiens interleukin 1 receptor, type I (IL1R1), mRNA NM_004633 Homo sapiens interleukin 1 receptor, type II (IL1R2), mRNA NM_016232 Homo sapiens interleukin 1 receptor-like 1 (IL1RL1), mRNA NM_003856 Homo sapiens interleukin 1 receptor-like 1 (IL1RL1), mRNA NM_003854 Homo sapiens interleukin 1 receptor-like 2 (IL1RL2), mRNA NM_000417 Homo sapiens interleukin 2 receptor, alpha (IL2RA), mRNA NM_000878 Homo sapiens interleukin 2 receptor, beta (IL2RB), mRNA NM_000206 Homo sapiens interleukin 2 receptor, gamma (severe combined immunodeficiency) (IL2RG), mRNA NM_002183 Homo sapiens interleukin 3 receptor, alpha (low affinity) (IL3RA), mRNA NM_000418 Homo sapiens interleukin 4 receptor (IL4R), mRNA NM_000564 Homo sapiens interleukin 5 receptor, alpha (IL5RA), mRNA NM_000565 Homo sapiens interleukin 6 receptor (IL6R), mRNA NM_002185 Homo sapiens interleukin 7 receptor (IL7R), mRNA NM_000634 Homo sapiens interleukin 8 receptor, alpha (IL8RA), mRNA NM_001557 Homo sapiens interleukin 8 receptor, beta (IL8RB), mRNA NM_002186 Homo sapiens interleukin 9 receptor (IL9R), mRNA NM_001558 Homo sapiens interleukin 10 receptor, alpha (IL10RA), mRNA NM_000628 Homo sapiens interleukin 10 receptor, beta (IL10RB), mRNA NM_004512 Homo sapiens interleukin 11 receptor, alpha (IL11RA), mRNA NM_005535 Homo sapiens interleukin 12 receptor, beta 1 (IL12RB1), mRNA NM_001559 Homo sapiens interleukin 12 receptor, beta 2 (IL12RB2), mRNA NM_001560 Homo sapiens interleukin 13 receptor, alpha 1 (IL13RA1), mRNA NM_000640 Homo sapiens interleukin 13 receptor, alpha 2 (IL13RA2), mRNA NM_002189 Homo sapiens interleukin 15 receptor, alpha (IL15RA), mRNA NM_014339 Homo sapiens interleukin 17 receptor (IL17R), mRNA NM_032732 Homo sapiens interleukin 17 receptor C (IL-17RC), mRNA NM_144640 Homo sapiens interleukin 17 receptor E (IL-17RE), mRNA NM_018725 Homo sapiens interleukin 17B receptor (IL17BR), mRNA NM_003855 Homo sapiens interleukin 18 receptor 1 (IL18R1), mRNA NM_003853 Homo sapiens interleukin 18 receptor accessory protein (IL18RAP), mRNA NM_014432 Homo sapiens interleukin 20 receptor, alpha (IL20RA), mRNA NM_021798 Homo sapiens interleukin 21 receptor (IL21 antisenseR), mRNA NM_021258 Homo sapiens interleukin 22 receptor (IL22R), mRNA NM_144701 Homo sapiens interleukin 23 receptor (IL23R), mRNA Interleukin Associated Proteins NM_004514 Homo sapiens interleukin enhancer binding factor 1 (ILF1), mRNA NM_004515 Homo sapiens interleukin enhancer binding factor 2, 45 kD (ILF2), mRNA NM_012218 Homo sapiens interleukin enhancer binding factor 3, 90 kD (ILF3), mRNA NM_004516 Homo sapiens interleukin enhancer binding factor 3, 90 kD (ILF3), mRNA NM_016123 Homo sapiens interleukin-1 receptor associated kinase 4 (IRAK4), mRNA NM_001569 Homo sapiens interleukin-1 receptor-associated kinase 1 (IRAK1), mRNA NM_001570 Homo sapiens interleukin-1 receptor-associated kinase 2 (IRAK2), mRNA NM_007199 Homo sapiens interleukin-1 receptor-associated kinase 3 (IRAK3), mRNA NM_134470 Homo sapiens interleukin 1 receptor accessory protein (IL1RAP), mRNA NM_002182 Homo sapiens interleukin 1 receptor accessory protein (IL1RAP), mRNA NM_014271 Homo sapiens interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), mRNA NM_017416 Homo sapiens interleukin 1 receptor accessory protein-like 2 (IL1RAPL2), mRNA NM_000577 Homo sapiens interleukin 1 receptor antagonist (IL1RN), mRNA NM_002184 Homo sapiens interleukin 6 signal transducer (gp130, oncostatin M receptor) (IL6ST), mRNA NM_005699 Homo sapiens interleukin 18 binding protein (IL18BP), mRNA

TABLE II Interleukin and Interleukin receptor siNA and Target Sequences Seq Seq Seq Pos Seq ID UPos Upper seq ID LPos Lower seq ID IL2RG NM_000206 3 AGAGCAAGCGCCAUGUUGA 1 3 AGAGCAAGCGCCAUGUUGA 1 25 UCAACAUGGCGCUUGCUCU 82 21 AAGCCAUCAUUACCAUUCA 2 21 AAGCCAUCAUUACCAUUCA 2 43 UGAAUGGUAAUGAUGGCUU 83 39 ACAUCCCUCUUAUUCCUGC 3 39 ACAUCCCUCUUAUUCCUGC 3 61 GCAGGAAUAAGAGGGAUGU 84 57 CAGCUGCCCCUGCUGGGAG 4 57 CAGCUGCCCCUGCUGGGAG 4 79 CUCCCAGCAGGGGCAGCUG 85 75 GUGGGGCUGAACACGACAA 5 75 GUGGGGCUGAACACGACAA 5 97 UUGUCGUGUUCAGCCCCAC 86 93 AUUCUGACGCCCAAUGGGA 6 93 AUUCUGACGCCCAAUGGGA 6 115 UCCCAUUGGGCGUCAGAAU 87 111 AAUGAAGACACCACAGCUG 7 111 AAUGAAGACACCACAGCUG 7 133 CAGCUGUGGUGUCUUCAUU 88 129 GAUUUCUUCCUGACCACUA 8 129 GAUUUCUUCCUGACCACUA 8 151 UAGUGGUCAGGAAGAAAUC 89 147 AUGCCCACUGACUCCCUCA 9 147 AUGCCCACUGACUCCCUCA 9 169 UGAGGGAGUCAGUGGGCAU 90 165 AGUGUUUCCACUCUGCCCC 10 165 AGUGUUUCCACUCUGCCCC 10 187 GGGGCAGAGUGGAAACACU 91 183 CUCCCAGAGGUUCAGUGUU 11 183 CUCCCAGAGGUUCAGUGUU 11 205 AACACUGAACCUCUGGGAG 92 201 UUUGUGUUCAAUGUCGAGU 12 201 UUUGUGUUCAAUGUCGAGU 12 223 ACUCGACAUUGAACACAAA 93 219 UACAUGAAUUGCACUUGGA 13 219 UACAUGAAUUGCACUUGGA 13 241 UCCAAGUGCAAUUCAUGUA 94 237 AACAGCAGCUCUGAGCCCC 14 237 AACAGCAGCUCUGAGCCCC 14 259 GGGGCUCAGAGCUGCUGUU 95 255 CAGCCUACCAACCUCACUC 15 255 CAGCCUACCAACCUCACUC 15 277 GAGUGAGGUUGGUAGGCUG 96 273 CUGCAUUAUUGGUACAAGA 16 273 CUGCAUUAUUGGUACAAGA 16 295 UCUUGUACCAAUAAUGCAG 97 291 AACUCGGAUAAUGAUAAAG 17 291 AACUCGGAUAAUGAUAAAG 17 313 CUUUAUCAUUAUCCGAGUU 98 309 GUCCAGAAGUGCAGCCACU 18 309 GUCCAGAAGUGCAGCCACU 18 331 AGUGGCUGCACUUCUGGAC 99 327 UAUCUAUUCUCUGAAGAAA 19 327 UAUCUAUUCUCUGAAGAAA 19 349 UUUCUUCAGAGAAUAGAUA 100 345 AUCACUUCUGGCUGUCAGU 20 345 AUCACUUCUGGCUGUCAGU 20 367 ACUGACAGCCAGAAGUGAU 101 363 UUGCAAAAAAAGGAGAUCC 21 363 UUGCAAAAAAAGGAGAUCC 21 385 GGAUCUCCUUUUUUUGCAA 102 381 CACCUCUACCAAACAUUUG 22 381 CACCUCUACCAAACAUUUG 22 403 CAAAUGUUUGGUAGAGGUG 103 399 GUUGUUCAGCUCCAGGACC 23 399 GUUGUUCAGCUCCAGGACC 23 421 GGUCCUGGAGCUGAACAAC 104 417 CCACGGGAACCCAGGAGAC 24 417 CCACGGGAACCCAGGAGAC 24 439 GUCUCCUGGGUUCCCGUGG 105 435 CAGGCCACACAGAUGCUAA 25 435 CAGGCCACACAGAUGCUAA 25 457 UUAGCAUCUGUGUGGCCUG 106 453 AAACUGCAGAAUCUGGUGA 26 453 AAACUGCAGAAUCUGGUGA 26 475 UCACCAGAUUCUGCAGUUU 107 471 AUCCCCUGGGCUCCAGAGA 27 471 AUCCCCUGGGCUCCAGAGA 27 493 UCUCUGGAGCCCAGGGGAU 108 489 AACCUAACACUUCACAAAC 28 489 AACCUAACACUUCACAAAC 28 511 GUUUGUGAAGUGUUAGGUU 109 507 CUGAGUGAAUCCCAGCUAG 29 507 CUGAGUGAAUCCCAGCUAG 29 529 CUAGCUGGGAUUCACUCAG 110 525 GAACUGAACUGGAACAACA 30 525 GAACUGAACUGGAACAACA 30 547 UGUUGUUCCAGUUCAGUUC 111 543 AGAUUCUUGAACCACUGUU 31 543 AGAUUCUUGAACCACUGUU 31 565 AACAGUGGUUCAAGAAUCU 112 561 UUGGAGCACUUGGUGCAGU 32 561 UUGGAGCACUUGGUGCAGU 32 583 ACUGCACCAAGUGCUCCAA 113 579 UACCGGACUGACUGGGACC 33 579 UACCGGACUGACUGGGACC 33 601 GGUCCCAGUCAGUCCGGUA 114 597 CACAGCUGGACUGAACAAU 34 597 CACAGCUGGACUGAACAAU 34 619 AUUGUUCAGUCCAGCUGUG 115 615 UCAGUGGAUUAUAGACAUA 35 615 UCAGUGGAUUAUAGACAUA 35 637 UAUGUCUAUAAUCCACUGA 116 633 AAGUUCUCCUUGCCUAGUG 36 633 AAGUUCUCCUUGCCUAGUG 36 655 CACUAGGCAAGGAGAACUU 117 651 GUGGAUGGGCAGAAACGCU 37 651 GUGGAUGGGCAGAAACGCU 37 673 AGCGUUUCUGCCCAUCCAC 118 669 UACACGUUUCGUGUUCGGA 38 669 UACACGUUUCGUGUUCGGA 38 691 UCCGAACACGAAACGUGUA 119 687 AGCCGCUUUAACCCACUCU 39 687 AGCCGCUUUAACCCACUCU 39 709 AGAGUGGGUUAAAGCGGCU 120 705 UGUGGAAGUGCUCAGCAUU 40 705 UGUGGAAGUGCUCAGCAUU 40 727 AAUGCUGAGCACUUCCACA 121 723 UGGAGUGAAUGGAGCCACC 41 723 UGGAGUGAAUGGAGCCACC 41 745 GGUGGCUCCAUUCACUCCA 122 741 CCAAUCCACUGGGGGAGCA 42 741 CCAAUCCACUGGGGGAGCA 42 763 UGCUCCCCCAGUGGAUUGG 123 759 AAUACUUCAAAAGAGAAUC 43 759 AAUACUUCAAAAGAGAAUC 43 781 GAUUCUCUUUUGAAGUAUU 124 777 CCUUUCCUGUUUGCAUUGG 44 777 CCUUUCCUGUUUGCAUUGG 44 799 CCAAUGCAAACAGGAAAGG 125 795 GAAGCCGUGGUUAUCUCUG 45 795 GAAGCCGUGGUUAUCUCUG 45 817 CAGAGAUAACCACGGCUUC 126 813 GUUGGCUCCAUGGGAUUGA 46 813 GUUGGCUCCAUGGGAUUGA 46 835 UCAAUCCCAUGGAGCCAAC 127 831 AUUAUCAGCCUUCUCUGUG 47 831 AUUAUCAGCCUUCUCUGUG 47 853 CACAGAGAAGGCUGAUAAU 128 849 GUGUAUUUCUGGCUGGAAC 48 849 GUGUAUUUCUGGCUGGAAC 48 871 GUUCCAGCCAGAAAUACAC 129 867 CGGACGAUGCCCCGAAUUC 49 867 CGGACGAUGCCCCGAAUUC 49 889 GAAUUCGGGGCAUCGUCCG 130 885 CCCACCCUGAAGAACCUAG 50 885 CCCACCCUGAAGAACCUAG 50 907 CUAGGUUCUUCAGGGUGGG 131 903 GAGGAUCUUGUUACUGAAU 51 903 GAGGAUCUUGUUACUGAAU 51 925 AUUCAGUAACAAGAUCCUC 132 921 UACCACGGGAACUUUUCGG 52 921 UACCACGGGAACUUUUCGG 52 943 CCGAAAAGUUCCCGUGGUA 133 939 GCCUGGAGUGGUGUGUCUA 53 939 GCCUGGAGUGGUGUGUCUA 53 961 UAGACACACCACUCCAGGC 134 957 AAGGGACUGGCUGAGAGUC 54 957 AAGGGACUGGCUGAGAGUC 54 979 GACUCUCAGCCAGUCCCUU 135 975 CUGCAGCCAGACUACAGUG 55 975 CUGCAGCCAGACUACAGUG 55 997 CACUGUAGUCUGGCUGCAG 136 993 GAACGACUCUGCCUCGUCA 56 993 GAACGACUCUGCCUCGUCA 56 1015 UGACGAGGCAGAGUCGUUC 137 1011 AGUGAGAUUCCCCCAAAAG 57 1011 AGUGAGAUUCCCCCAAAAG 57 1033 CUUUUGGGGGAAUCUCACU 138 1029 GGAGGGGCCCUUGGGGAGG 58 1029 GGAGGGGCCCUUGGGGAGG 58 1051 CCUCCCCAAGGGCCCCUCC 139 1047 GGGCCUGGGGCCUCCCCAU 59 1047 GGGCCUGGGGCCUCCCCAU 59 1069 AUGGGGAGGCCCCAGGCCC 140 1065 UGCAACCAGCAUAGCCCCU 60 1065 UGCAACCAGCAUAGCCCCU 60 1087 AGGGGCUAUGCUGGUUGCA 141 1083 UACUGGGCCCCCCCAUGUU 61 1083 UACUGGGCCCCCCCAUGUU 61 1105 AACAUGGGGGGGCCCAGUA 142 1101 UACACCCUAAAGCCUGAAA 62 1101 UACACCCUAAAGCCUGAAA 62 1123 UUUCAGGCUUUAGGGUGUA 143 1119 ACCUGAACCCCAAUCCUCU 63 1119 ACCUGAACCCCAAUCCUCU 63 1141 AGAGGAUUGGGGUUCAGGU 144 1137 UGACAGAAGAACCCCAGGG 64 1137 UGACAGAAGAACCCCAGGG 64 1159 CCCUGGGGUUCUUCUGUCA 145 1155 GUCCUGUAGCCCUAAGUGG 65 1155 GUCCUGUAGCCCUAAGUGG 65 1177 CCACUUAGGGCUACAGGAC 146 1173 GUACUAACUUUCCUUCAUU 66 1173 GUACUAACUUUCCUUCAUU 66 1195 AAUGAAGGAAAGUUAGUAC 147 1191 UCAACCCACCUGCGUCUCA 67 1191 UCAACCCACCUGCGUCUCA 67 1213 UGAGACGCAGGUGGGUUGA 148 1209 AUACUCACCUCACCCCACU 68 1209 AUACUCACCUCACCCCACU 68 1231 AGUGGGGUGAGGUGAGUAU 149 1227 UGUGGCUGAUUUGGAAUUU 69 1227 UGUGGCUGAUUUGGAAUUU 69 1249 AAAUUCCAAAUCAGCCACA 150 1245 UUGUGCCCCCAUGUAAGCA 70 1245 UUGUGCCCCCAUGUAAGCA 70 1267 UGCUUACAUGGGGGCACAA 151 1263 ACCCCUUCAUUUGGCAUUC 71 1263 ACCCCUUCAUUUGGCAUUC 71 1285 GAAUGCCAAAUGAAGGGGU 152 1281 CCCCACUUGAGAAUUACCC 72 1281 CCCCACUUGAGAAUUACCC 72 1303 GGGUAAUUCUCAAGUGGGG 153 1299 CUUUUGCCCCGAACAUGUU 73 1299 CUUUUGCCCCGAACAUGUU 73 1321 AACAUGUUCGGGGCAAAAG 154 1317 UUUUCUUCUCCCUCAGUCU 74 1317 UUUUCUUCUCCCUCAGUCU 74 1339 AGACUGAGGGAGAAGAAAA 155 1335 UGGCCCUUCCUUUUCGCAG 75 1335 UGGCCCUUCCUUUUCGCAG 75 1357 CUGCGAAAAGGAAGGGCCA 156 1353 GGAUUCUUCCUCCCUCCCU 76 1353 GGAUUCUUCCUCCCUCCCU 76 1375 AGGGAGGGAGGAAGAAUCC 157 1371 UCUUUCCCUCCCUUCCUCU 77 1371 UCUUUCCCUCCCUUCCUCU 77 1393 AGAGGAAGGGAGGGAAAGA 158 1389 UUUCCAUCUACCCUCCGAU 78 1389 UUUCCAUCUACCCUCCGAU 78 1411 AUCGGAGGGUAGAUGGAAA 159 1407 UUGUUCCUGAACCGAUGAG 79 1407 UUGUUCCUGAACCGAUGAG 79 1429 CUCAUCGGUUCAGGAACAA 160 1425 GAAAUAAAGUUUCUGUUGA 80 1425 GAAAUAAAGUUUCUGUUGA 80 1447 UCAACAGAAACUUUAUUUC 161 1431 AAGUUUCUGUUGAUAAUCA 81 1431 AAGUUUCUGUUGAUAAUCA 81 1453 UGAUUAUCAACAGAAACUU 162 IL4 NM_000589 3 CUAUGCAAAGCAAAAAGCC 163 3 CUAUGCAAAGCAAAAAGCC 163 25 GGCUUUUUGCUUUGCAUAG 214 21 CAGCAGCAGCCCCAAGCUG 164 21 CAGCAGCAGCCCCAAGCUG 164 43 CAGCUUGGGGCUGCUGCUG 215 39 GAUAAGAUUAAUCUAAAGA 165 39 GAUAAGAUUAAUCUAAAGA 165 61 UCUUUAGAUUAAUCUUAUC 216 57 AGCAAAUUAUGGUGUAAUU 166 57 AGCAAAUUAUGGUGUAAUU 166 79 AAUUACACCAUAAUUUGCU 217 75 UUCCUAUGCUGAAACUUUG 167 75 UUCCUAUGCUGAAACUUUG 167 97 CAAAGUUUCAGCAUAGGAA 218 93 GUAGUUAAUUUUUUAAAAA 168 93 GUAGUUAAUUUUUUAAAAA 168 115 UUUUUAAAAAAUUAACUAC 219 111 AGGUUUCAUUUUCCUAUUG 169 111 AGGUUUCAUUUUCCUAUUG 169 133 CAAUAGGAAAAUGAAACCU 220 129 GGUCUGAUUUCACAGGAAC 170 129 GGUCUGAUUUCACAGGAAC 170 151 GUUCCUGUGAAAUCAGACC 221 147 CAUUUUACCUGUUUGUGAG 171 147 CAUUUUACCUGUUUGUGAG 171 169 CUCACAAACAGGUAAAAUG 222 165 GGCAUUUUUUCUCCUGGAA 172 165 GGCAUUUUUUCUCCUGGAA 172 187 UUCCAGGAGAAAAAAUGCC 223 183 AGAGAGGUGCUGAUUGGCC 173 183 AGAGAGGUGCUGAUUGGCC 173 205 GGCCAAUCAGCACCUCUCU 224 201 CCCAAGUGACUGACAAUCU 174 201 CCCAAGUGACUGACAAUCU 174 223 AGAUUGUCAGUCACUUGGG 225 219 UGGUGUAACGAAAAUUUCC 175 219 UGGUGUAACGAAAAUUUCC 175 241 GGAAAUUUUCGUUACACCA 226 237 CAAUGUAAACUCAUUUUCC 176 237 CAAUGUAAACUCAUUUUCC 176 259 GGAAAAUGAGUUUACAUUG 227 255 CCUCGGUUUCAGCAAUUUU 177 255 CCUCGGUUUCAGCAAUUUU 177 277 AAAAUUGCUGAAACCGAGG 228 273 UAAAUCUAUAUAUAGAGAU 178 273 UAAAUCUAUAUAUAGAGAU 178 295 AUCUCUAUAUAUAGAUUUA 229 291 UAUCUUUGUCAGCAUUGCA 179 291 UAUCUUUGUCAGCAUUGCA 179 313 UGCAAUGCUGACAAAGAUA 230 309 AUCGUUAGCUUCUCCUGAU 180 309 AUCGUUAGCUUCUCCUGAU 180 331 AUCAGGAGAAGCUAACGAU 231 327 UAAACUAAUUGCCUCACAU 181 327 UAAACUAAUUGCCUCACAU 181 349 AUGUGAGGCAAUUAGUUUA 232 345 UUGUCACUGCAAAUCGACA 182 345 UUGUCACUGCAAAUCGACA 182 367 UGUCGAUUUGCAGUGACAA 233 363 ACCUAUUAAUGGGUCUCAC 183 363 ACCUAUUAAUGGGUCUCAC 183 385 GUGAGACCCAUUAAUAGGU 234 381 CCUCCCAACUGCUUCCCCC 184 381 CCUCCCAACUGCUUCCCCC 184 403 GGGGGAAGCAGUUGGGAGG 235 399 CUCUGUUCUUCCUGCUAGC 185 399 CUCUGUUCUUCCUGCUAGC 185 421 GCUAGCAGGAAGAACAGAG 236 417 CAUGUGCCGGCAACUUUGU 186 417 CAUGUGCCGGCAACUUUGU 186 439 ACAAAGUUGCCGGCACAUG 237 435 UCCACGGACACAAGUGCGA 187 435 UCCACGGACACAAGUGCGA 187 457 UCGCACUUGUGUCCGUGGA 238 453 AUAUCACCUUACAGGAGAU 188 453 AUAUCACCUUACAGGAGAU 188 475 AUCUCCUGUAAGGUGAUAU 239 471 UCAUCAAAACUUUGAACAG 189 471 UCAUCAAAACUUUGAACAG 189 493 CUGUUCAAAGUUUUGAUGA 240 489 GCCUCACAGAGCAGAAGAC 190 489 GCCUCACAGAGCAGAAGAC 190 511 GUCUUCUGCUCUGUGAGGC 241 507 CUCUGUGCACCGAGUUGAC 191 507 CUCUGUGCACCGAGUUGAC 191 529 GUCAACUCGGUGCACAGAG 242 525 CCGUAACAGACAUCUUUGC 192 525 CCGUAACAGACAUCUUUGC 192 547 GCAAAGAUGUCUGUUACGG 243 543 CUGCCUCCAAGAACACAAC 193 543 CUGCCUCCAAGAACACAAC 193 565 GUUGUGUUCUUGGAGGCAG 244 561 CUGAGAAGGAAACCUUCUG 194 561 CUGAGAAGGAAACCUUCUG 194 583 CAGAAGGUUUCCUUCUCAG 245 579 GCAGGGCUGCGACUGUGCU 195 579 GCAGGGCUGCGACUGUGCU 195 601 AGCACAGUCGCAGCCCUGC 246 597 UCCGGCAGUUCUACAGCCA 196 597 UCCGGCAGUUCUACAGCCA 196 619 UGGCUGUAGAACUGCCGGA 247 615 ACCAUGAGAAGGACACUCG 197 615 ACCAUGAGAAGGACACUCG 197 637 CGAGUGUCCUUCUCAUGGU 248 633 GCUGCCUGGGUGCGACUGC 198 633 GCUGCCUGGGUGCGACUGC 198 655 GCAGUCGCACCCAGGCAGC 249 651 CACAGCAGUUCCACAGGCA 199 651 CACAGCAGUUCCACAGGCA 199 673 UGCCUGUGGAACUGCUGUG 250 669 ACAAGCAGCUGAUCCGAUU 200 669 ACAAGCAGCUGAUCCGAUU 200 691 AAUCGGAUCAGCUGCUUGU 251 687 UCCUGAAACGGCUCGACAG 201 687 UCCUGAAACGGCUCGACAG 201 709 CUGUCGAGCCGUUUCAGGA 252 705 GGAACCUCUGGGGCCUGGC 202 705 GGAACCUCUGGGGCCUGGC 202 727 GCCAGGCCCCAGAGGUUCC 253 723 CGGGCUUGAAUUCCUGUCC 203 723 CGGGCUUGAAUUCCUGUCC 203 745 GGACAGGAAUUCAAGCCCG 254 741 CUGUGAAGGAAGCCAACCA 204 741 CUGUGAAGGAAGCCAACCA 204 763 UGGUUGGCUUCCUUCACAG 255 759 AGAGUACGUUGGAAAACUU 205 759 AGAGUACGUUGGAAAACUU 205 781 AAGUUUUCCAACGUACUCU 256 777 UCUUGGAAAGGCUAAAGAC 206 777 UCUUGGAAAGGCUAAAGAC 206 799 GUCUUUAGCCUUUCCAAGA 257 795 CGAUCAUGAGAGAGAAAUA 207 795 CGAUCAUGAGAGAGAAAUA 207 817 UAUUUCUCUCUCAUGAUCG 258 813 AUUCAAAGUGUUCGAGCUG 208 813 AUUCAAAGUGUUCGAGCUG 208 835 CAGCUCGAACACUUUGAAU 259 831 GAAUAUUUUAAUUUAUGAG 209 831 GAAUAUUUUAAUUUAUGAG 209 853 CUCAUAAAUUAAAAUAUUC 260 849 GUUUUUGAUAGCUUUAUUU 210 849 GUUUUUGAUAGCUUUAUUU 210 871 AAAUAAAGCUAUCAAAAAC 261 867 UUUUAAGUAUUUAUAUAUU 211 867 UUUUAAGUAUUUAUAUAUU 211 889 AAUAUAUAAAUACUUAAAA 262 885 UUAUAACUCAUCAUAAAAU 212 885 UUAUAACUCAUCAUAAAAU 212 907 AUUUUAUGAUGAGUUAUAA 263 901 AAUAAAGUAUAUAUAGAAU 213 901 AAUAAAGUAUAUAUAGAAU 213 923 AUUCUAUAUAUACUUUAUU 264 IL4R NM_000418 3 CGAAUGGAGCAGGGGCGCG 265 3 CGAAUGGAGCAGGGGCGCG 265 25 CGCGCCCCUGCUCCAUUCG 465 21 GCAGAUAAUUAAAGAUUUA 266 21 GCAGAUAAUUAAAGAUUUA 266 43 UAAAUCUUUAAUUAUCUGC 466 39 ACACACAGCUGGAAGAAAU 267 39 ACACACAGCUGGAAGAAAU 267 61 AUUUCUUCCAGCUGUGUGU 467 57 UCAUAGAGAAGCCGGGCGU 268 57 UCAUAGAGAAGCCGGGCGU 268 79 ACGCCCGGCUUCUCUAUGA 468 75 UGGUGGCUCAUGCCUAUAA 269 75 UGGUGGCUCAUGCCUAUAA 269 97 UUAUAGGCAUGAGCCACCA 469 93 AUCCCAGCACUUUUGGAGG 270 93 AUCCCAGCACUUUUGGAGG 270 115 CCUCCAAAAGUGCUGGGAU 470 111 GCUGAGGCGGGCAGAUCAC 271 111 GCUGAGGCGGGCAGAUCAC 271 133 GUGAUCUGCCCGCCUCAGC 471 129 CUUGAGAUCAGGAGUUCGA 272 129 CUUGAGAUCAGGAGUUCGA 272 151 UCGAACUCCUGAUCUCAAG 472 147 AGACCAGCCUGGUGCCUUG 273 147 AGACCAGCCUGGUGCCUUG 273 169 CAAGGCACCAGGCUGGUCU 473 165 GGCAUCUCCCAAUGGGGUG 274 165 GGCAUCUCCCAAUGGGGUG 274 187 CACCCCAUUGGGAGAUGCC 474 183 GGCUUUGCUCUGGGCUCCU 275 183 GGCUUUGCUCUGGGCUCCU 275 205 AGGAGCCCAGAGCAAAGCC 475 201 UGUUCCCUGUGAGCUGCCU 276 201 UGUUCCCUGUGAGCUGCCU 276 223 AGGCAGCUCACAGGGAACA 476 219 UGGUCCUGCUGCAGGUGGC 277 219 UGGUCCUGCUGCAGGUGGC 277 241 GCCACCUGCAGCAGGACCA 477 237 CAAGCUCUGGGAACAUGAA 278 237 CAAGCUCUGGGAACAUGAA 278 259 UUCAUGUUCCCAGAGCUUG 478 255 AGGUCUUGCAGGAGCCCAC 279 255 AGGUCUUGCAGGAGCCCAC 279 277 GUGGGCUCCUGCAAGACCU 479 273 CCUGCGUCUCCGACUACAU 280 273 CCUGCGUCUCCGACUACAU 280 295 AUGUAGUCGGAGACGCAGG 480 291 UGAGCAUCUCUACUUGCGA 281 291 UGAGCAUCUCUACUUGCGA 281 313 UCGCAAGUAGAGAUGCUCA 481 309 AGUGGAAGAUGAAUGGUCC 282 309 AGUGGAAGAUGAAUGGUCC 282 331 GGACCAUUCAUCUUCCACU 482 327 CCACCAAUUGCAGCACCGA 283 327 CCACCAAUUGCAGCACCGA 283 349 UCGGUGCUGCAAUUGGUGG 483 345 AGCUCCGCCUGUUGUACCA 284 345 AGCUCCGCCUGUUGUACCA 284 367 UGGUACAACAGGCGGAGCU 484 363 AGCUGGUUUUUCUGCUCUC 285 363 AGCUGGUUUUUCUGCUCUC 285 385 GAGAGCAGAAAAACCAGCU 485 381 CCGAAGCCCACACGUGUAU 286 381 CCGAAGCCCACACGUGUAU 286 403 AUACACGUGUGGGCUUCGG 486 399 UCCCUGAGAACAACGGAGG 287 399 UCCCUGAGAACAACGGAGG 287 421 CCUCCGUUGUUCUCAGGGA 487 417 GCGCGGGGUGCGUGUGCCA 288 417 GCGCGGGGUGCGUGUGCCA 288 439 UGGCACACGCACCCCGCGC 488 435 ACCUGCUCAUGGAUGACGU 289 435 ACCUGCUCAUGGAUGACGU 289 457 ACGUCAUCCAUGAGCAGGU 489 453 UGGUCAGUGCGGAUAACUA 290 453 UGGUCAGUGCGGAUAACUA 290 475 UAGUUAUCCGCACUGACCA 490 471 AUACACUGGACCUGUGGGC 291 471 AUACACUGGACCUGUGGGC 291 493 GCCCACAGGUCCAGUGUAU 491 489 CUGGGCAGCAGCUGCUGUG 292 489 CUGGGCAGCAGCUGCUGUG 292 511 CACAGCAGCUGCUGCCCAG 492 507 GGAAGGGCUCCUUCAAGCC 293 507 GGAAGGGCUCCUUCAAGCC 293 529 GGCUUGAAGGAGCCCUUCC 493 525 CCAGCGAGCAUGUGAAACC 294 525 CCAGCGAGCAUGUGAAACC 294 547 GGUUUCACAUGCUCGCUGG 494 543 CCAGGGCCCCAGGAAACCU 295 543 CCAGGGCCCCAGGAAACCU 295 565 AGGUUUCCUGGGGCCCUGG 495 561 UGACAGUUCACACCAAUGU 296 561 UGACAGUUCACACCAAUGU 296 583 ACAUUGGUGUGAACUGUCA 496 579 UCUCCGACACUCUGCUGCU 297 579 UCUCCGACACUCUGCUGCU 297 601 AGCAGCAGAGUGUCGGAGA 497 597 UGACCUGGAGCAACCCGUA 298 597 UGACCUGGAGCAACCCGUA 298 619 UACGGGUUGCUCCAGGUCA 498 615 AUCCCCCUGACAAUUACCU 299 615 AUCCCCCUGACAAUUACCU 299 637 AGGUAAUUGUCAGGGGGAU 499 633 UGUAUAAUCAUCUCACCUA 300 633 UGUAUAAUCAUCUCACCUA 300 655 UAGGUGAGAUGAUUAUACA 500 651 AUGCAGUCAACAUUUGGAG 301 651 AUGCAGUCAACAUUUGGAG 301 673 CUCCAAAUGUUGACUGCAU 501 669 GUGAAAACGACCCGGCAGA 302 669 GUGAAAACGACCCGGCAGA 302 691 UCUGCCGGGUCGUUUUCAC 502 687 AUUUCAGAAUCUAUAACGU 303 687 AUUUCAGAAUCUAUAACGU 303 709 ACGUUAUAGAUUCUGAAAU 503 705 UGACCUACCUAGAACCCUC 304 705 UGACCUACCUAGAACCCUC 304 727 GAGGGUUCUAGGUAGGUCA 504 723 CCCUCCGCAUCGCAGCCAG 305 723 CCCUCCGCAUCGCAGCCAG 305 745 CUGGCUGCGAUGCGGAGGG 505 741 GCACCCUGAAGUCUGGGAU 306 741 GCACCCUGAAGUCUGGGAU 306 763 AUCCCAGACUUCAGGGUGC 506 759 UUUCCUACAGGGCACGGGU 307 759 UUUCCUACAGGGCACGGGU 307 781 ACCCGUGCCCUGUAGGAAA 507 777 UGAGGGCCUGGGCUCAGUG 308 777 UGAGGGCCUGGGCUCAGUG 308 799 CACUGAGCCCAGGCCCUCA 508 795 GCUAUAACACCACCUGGAG 309 795 GCUAUAACACCACCUGGAG 309 817 CUCCAGGUGGUGUUAUAGC 509 813 GUGAGUGGAGCCCCAGCAC 310 813 GUGAGUGGAGCCCCAGCAC 310 835 GUGCUGGGGCUCCACUCAC 510 831 CCAAGUGGCACAACUCCUA 311 831 CCAAGUGGCACAACUCCUA 311 853 UAGGAGUUGUGCCACUUGG 511 849 ACAGGGAGCCCUUCGAGCA 312 849 ACAGGGAGCCCUUCGAGCA 312 871 UGCUCGAAGGGCUCCCUGU 512 867 AGCACCUCCUGCUGGGCGU 313 867 AGCACCUCCUGCUGGGCGU 313 889 ACGCCCAGCAGGAGGUGCU 513 885 UCAGCGUUUCCUGCAUUGU 314 885 UCAGCGUUUCCUGCAUUGU 314 907 ACAAUGCAGGAAACGCUGA 514 903 UCAUCCUGGCCGUCUGCCU 315 903 UCAUCCUGGCCGUCUGCCU 315 925 AGGCAGACGGCCAGGAUGA 515 921 UGUUGUGCUAUGUCAGCAU 316 921 UGUUGUGCUAUGUCAGCAU 316 943 AUGCUGACAUAGCACAACA 516 939 UCACCAAGAUUAAGAAAGA 317 939 UCACCAAGAUUAAGAAAGA 317 961 UCUUUCUUAAUCUUGGUGA 517 957 AAUGGUGGGAUCAGAUUCC 318 957 AAUGGUGGGAUCAGAUUCC 318 979 GGAAUCUGAUCCCACCAUU 518 975 CCAACCCAGCCCGCAGCCG 319 975 CCAACCCAGCCCGCAGCCG 319 997 CGGCUGCGGGCUGGGUUGG 519 993 GCCUCGUGGCUAUAAUAAU 320 993 GCCUCGUGGCUAUAAUAAU 320 1015 AUUAUUAUAGCCACGAGGC 520 1011 UCCAGGAUGCUCAGGGGUC 321 1011 UCCAGGAUGCUCAGGGGUC 321 1033 GACCCCUGAGCAUCCUGGA 521 1029 CACAGUGGGAGAAGCGGUC 322 1029 CACAGUGGGAGAAGCGGUC 322 1051 GACCGCUUCUCCCACUGUG 522 1047 CCCGAGGCCAGGAACCAGC 323 1047 CCCGAGGCCAGGAACCAGC 323 1069 GCUGGUUCCUGGCCUCGGG 523 1065 CCAAGUGCCCACACUGGAA 324 1065 CCAAGUGCCCACACUGGAA 324 1087 UUCCAGUGUGGGCACUUGG 524 1083 AGAAUUGUCUUACCAAGCU 325 1083 AGAAUUGUCUUACCAAGCU 325 1105 AGCUUGGUAAGACAAUUCU 525 1101 UCUUGCCCUGUUUUCUGGA 326 1101 UCUUGCCCUGUUUUCUGGA 326 1123 UCCAGAAAACAGGGCAAGA 526 1119 AGCACAACAUGAAAAGGGA 327 1119 AGCACAACAUGAAAAGGGA 327 1141 UCCCUUUUCAUGUUGUGCU 527 1137 AUGAAGAUCCUCACAAGGC 328 1137 AUGAAGAUCCUCACAAGGC 328 1159 GCCUUGUGAGGAUCUUCAU 528 1155 CUGCCAAAGAGAUGCCUUU 329 1155 CUGCCAAAGAGAUGCCUUU 329 1177 AAAGGCAUCUCUUUGGCAG 529 1173 UCCAGGGCUCUGGAAAAUC 330 1173 UCCAGGGCUCUGGAAAAUC 330 1195 GAUUUUCCAGAGCCCUGGA 530 1191 CAGCAUGGUGCCCAGUGGA 331 1191 CAGCAUGGUGCCCAGUGGA 331 1213 UCCACUGGGCACCAUGCUG 531 1209 AGAUCAGCAAGACAGUCCU 332 1209 AGAUCAGCAAGACAGUCCU 332 1231 AGGACUGUCUUGCUGAUCU 532 1227 UCUGGCCAGAGAGCAUCAG 333 1227 UCUGGCCAGAGAGCAUCAG 333 1249 CUGAUGCUCUCUGGCCAGA 533 1245 GCGUGGUGCGAUGUGUGGA 334 1245 GCGUGGUGCGAUGUGUGGA 334 1267 UCCACACAUCGCACCACGC 534 1263 AGUUGUUUGAGGCCCCGGU 335 1263 AGUUGUUUGAGGCCCCGGU 335 1285 ACCGGGGCCUCAAACAACU 535 1281 UGGAGUGUGAGGAGGAGGA 336 1281 UGGAGUGUGAGGAGGAGGA 336 1303 UCCUCCUCCUCACACUCCA 536 1299 AGGAGGUAGAGGAAGAAAA 337 1299 AGGAGGUAGAGGAAGAAAA 337 1321 UUUUCUUCCUCUACCUCCU 537 1317 AAGGGAGCUUCUGUGCAUC 338 1317 AAGGGAGCUUCUGUGCAUC 338 1339 GAUGCACAGAAGCUCCCUU 538 1335 CGCCUGAGAGCAGCAGGGA 339 1335 CGCCUGAGAGCAGCAGGGA 339 1357 UCCCUGCUGCUCUCAGGCG 539 1353 AUGACUUCCAGGAGGGAAG 340 1353 AUGACUUCCAGGAGGGAAG 340 1375 CUUCCCUCCUGGAAGUCAU 540 1371 GGGAGGGCAUUGUGGCCCG 341 1371 GGGAGGGCAUUGUGGCCCG 341 1393 CGGGCCACAAUGCCCUCCC 541 1389 GGCUAACAGAGAGCCUGUU 342 1389 GGCUAACAGAGAGCCUGUU 342 1411 AACAGGCUCUCUGUUAGCC 542 1407 UCCUGGACCUGCUCGGAGA 343 1407 UCCUGGACCUGCUCGGAGA 343 1429 UCUCCGAGCAGGUCCAGGA 543 1425 AGGAGAAUGGGGGCUUUUG 344 1425 AGGAGAAUGGGGGCUUUUG 344 1447 CAAAAGCCCCCAUUCUCCU 544 1443 GCCAGCAGGACAUGGGGGA 345 1443 GCCAGCAGGACAUGGGGGA 345 1465 UCCCCCAUGUCCUGCUGGC 545 1461 AGUCAUGCCUUCUUCCACC 346 1461 AGUCAUGCCUUCUUCCACC 346 1483 GGUGGAAGAAGGCAUGACU 546 1479 CUUCGGGAAGUACGAGUGC 347 1479 CUUCGGGAAGUACGAGUGC 347 1501 GCACUCGUACUUCCCGAAG 547 1497 CUCACAUGCCCUGGGAUGA 348 1497 CUCACAUGCCCUGGGAUGA 348 1519 UCAUCCCAGGGCAUGUGAG 548 1515 AGUUCCCAAGUGCAGGGCC 349 1515 AGUUCCCAAGUGCAGGGCC 349 1537 GGCCCUGCACUUGGGAACU 549 1533 CCAAGGAGGCACCUCCCUG 350 1533 CCAAGGAGGCACCUCCCUG 350 1555 CAGGGAGGUGCCUCCUUGG 550 1551 GGGGCAAGGAGCAGCCUCU 351 1551 GGGGCAAGGAGCAGCCUCU 351 1573 AGAGGCUGCUCCUUGCCCC 551 1569 UCCACCUGGAGCCAAGUCC 352 1569 UCCACCUGGAGCCAAGUCC 352 1591 GGACUUGGCUCCAGGUGGA 552 1587 CUCCUGCCAGCCCGACCCA 353 1587 CUCCUGCCAGCCCGACCCA 353 1609 UGGGUCGGGCUGGCAGGAG 553 1605 AGAGUCCAGACAACCUGAC 354 1605 AGAGUCCAGACAACCUGAC 354 1627 GUCAGGUUGUCUGGACUCU 554 1623 CUUGCACAGAGACGCCCCU 355 1623 CUUGCACAGAGACGCCCCU 355 1645 AGGGGCGUCUCUGUGCAAG 555 1641 UCGUCAUCGCAGGCAACCC 356 1641 UCGUCAUCGCAGGCAACCC 356 1663 GGGUUGCCUGCGAUGACGA 556 1659 CUGCUUACCGCAGCUUCAG 357 1659 CUGCUUACCGCAGCUUCAG 357 1681 CUGAAGCUGCGGUAAGCAG 557 1677 GCAACUCCCUGAGCCAGUC 358 1677 GCAACUCCCUGAGCCAGUC 358 1699 GACUGGCUCAGGGAGUUGC 558 1695 CACCGUGUCCCAGAGAGCU 359 1695 CACCGUGUCCCAGAGAGCU 359 1717 AGCUCUCUGGGACACGGUG 559 1713 UGGGUCCAGACCCACUGCU 360 1713 UGGGUCCAGACCCACUGCU 360 1735 AGCAGUGGGUCUGGACCCA 560 1731 UGGCCAGACACCUGGAGGA 361 1731 UGGCCAGACACCUGGAGGA 361 1753 UCCUCCAGGUGUCUGGCCA 561 1749 AAGUAGAACCCGAGAUGCC 362 1749 AAGUAGAACCCGAGAUGCC 362 1771 GGCAUCUCGGGUUCUACUU 562 1767 CCUGUGUCCCCCAGCUCUC 363 1767 CCUGUGUCCCCCAGCUCUC 363 1789 GAGAGCUGGGGGACACAGG 563 1785 CUGAGCCAACCACUGUGCC 364 1785 CUGAGCCAACCACUGUGCC 364 1807 GGCACAGUGGUUGGCUCAG 564 1803 CCCAACCUGAGCCAGAAAC 365 1803 CCCAACCUGAGCCAGAAAC 365 1825 GUUUCUGGCUCAGGUUGGG 565 1821 CCUGGGAGCAGAUCCUCCG 366 1821 CCUGGGAGCAGAUCCUCCG 366 1843 CGGAGGAUCUGCUCCCAGG 566 1839 GCCGAAAUGUCCUCCAGCA 367 1839 GCCGAAAUGUCCUCCAGCA 367 1861 UGCUGGAGGACAUUUCGGC 567 1857 AUGGGGCAGCUGCAGCCCC 368 1857 AUGGGGCAGCUGCAGCCCC 368 1879 GGGGCUGCAGCUGCCCCAU 568 1875 CCGUCUCGGCCCCCACCAG 369 1875 CCGUCUCGGCCCCCACCAG 369 1897 CUGGUGGGGGCCGAGACGG 569 1893 GUGGCUAUCAGGAGUUUGU 370 1893 GUGGCUAUCAGGAGUUUGU 370 1915 ACAAACUCCUGAUAGCCAC 570 1911 UACAUGCGGUGGAGCAGGG 371 1911 UACAUGCGGUGGAGCAGGG 371 1933 CCCUGCUCCACCGCAUGUA 571 1929 GUGGCACCCAGGCCAGUGC 372 1929 GUGGCACCCAGGCCAGUGC 372 1951 GCACUGGCCUGGGUGCCAC 572 1947 CGGUGGUGGGCUUGGGUCC 373 1947 CGGUGGUGGGCUUGGGUCC 373 1969 GGACCCAAGCCCACCACCG 573 1965 CCCCAGGAGAGGCUGGUUA 374 1965 CCCCAGGAGAGGCUGGUUA 374 1987 UAACCAGCCUCUCCUGGGG 574 1983 ACAAGGCCUUCUCAAGCCU 375 1983 ACAAGGCCUUCUCAAGCCU 375 2005 AGGCUUGAGAAGGCCUUGU 575 2001 UGCUUGCCAGCAGUGCUGU 376 2001 UGCUUGCCAGCAGUGCUGU 376 2023 ACAGCACUGCUGGCAAGCA 576 2019 UGUCCCCAGAGAAAUGUGG 377 2019 UGUCCCCAGAGAAAUGUGG 377 2041 CCACAUUUCUCUGGGGACA 577 2037 GGUUUGGGGCUAGCAGUGG 378 2037 GGUUUGGGGCUAGCAGUGG 378 2059 CCACUGCUAGCCCCAAACC 578 2055 GGGAAGAGGGGUAUAAGCC 379 2055 GGGAAGAGGGGUAUAAGCC 379 2077 GGCUUAUACCCCUCUUCCC 579 2073 CUUUCCAAGACCUCAUUCC 380 2073 CUUUCCAAGACCUCAUUCC 380 2095 GGAAUGAGGUCUUGGAAAG 580 2091 CUGGCUGCCCUGGGGACCC 381 2091 CUGGCUGCCCUGGGGACCC 381 2113 GGGUCCCCAGGGCAGCCAG 581 2109 CUGCCCCAGUCCCUGUCCC 382 2109 CUGCCCCAGUCCCUGUCCC 382 2131 GGGACAGGGACUGGGGCAG 582 2127 CCUUGUUCACCUUUGGACU 383 2127 CCUUGUUCACCUUUGGACU 383 2149 AGUCCAAAGGUGAACAAGG 583 2145 UGGACAGGGAGCCACCUCG 384 2145 UGGACAGGGAGCCACCUCG 384 2167 CGAGGUGGCUCCCUGUCCA 584 2163 GCAGUCCGCAGAGCUCACA 385 2163 GCAGUCCGCAGAGCUCACA 385 2185 UGUGAGCUCUGCGGACUGC 585 2181 AUCUCCCAAGCAGCUCCCC 386 2181 AUCUCCCAAGCAGCUCCCC 386 2203 GGGGAGCUGCUUGGGAGAU 586 2199 CAGAGCACCUGGGUCUGGA 387 2199 CAGAGCACCUGGGUCUGGA 387 2221 UCCAGACCCAGGUGCUCUG 587 2217 AGCCGGGGGAAAAGGUAGA 388 2217 AGCCGGGGGAAAAGGUAGA 388 2239 UCUACCUUUUCCCCCGGCU 588 2235 AGGACAUGCCAAAGCCCCC 389 2235 AGGACAUGCCAAAGCCCCC 389 2257 GGGGGCUUUGGCAUGUCCU 589 2253 CACUUCCCCAGGAGCAGGC 390 2253 CACUUCCCCAGGAGCAGGC 390 2275 GCCUGCUCCUGGGGAAGUG 590 2271 CCACAGACCCCCUUGUGGA 391 2271 CCACAGACCCCCUUGUGGA 391 2293 UCCACAAGGGGGUCUGUGG 591 2289 ACAGCCUGGGCAGUGGCAU 392 2289 ACAGCCUGGGCAGUGGCAU 392 2311 AUGCCACUGCCCAGGCUGU 592 2307 UUGUCUACUCAGCCCUUAC 393 2307 UUGUCUACUCAGCCCUUAC 393 2329 GUAAGGGCUGAGUAGACAA 593 2325 CCUGCCACCUGUGCGGCCA 394 2325 CCUGCCACCUGUGCGGCCA 394 2347 UGGCCGCACAGGUGGCAGG 594 2343 ACCUGAAACAGUGUCAUGG 395 2343 ACCUGAAACAGUGUCAUGG 395 2365 CCAUGACACUGUUUCAGGU 595 2361 GCCAGGAGGAUGGUGGCCA 396 2361 GCCAGGAGGAUGGUGGCCA 396 2383 UGGCCACCAUCCUCCUGGC 596 2379 AGACCCCUGUCAUGGCCAG 397 2379 AGACCCCUGUCAUGGCCAG 397 2401 CUGGCCAUGACAGGGGUCU 597 2397 GUCCUUGCUGUGGCUGCUG 398 2397 GUCCUUGCUGUGGCUGCUG 398 2419 CAGCAGCCACAGCAAGGAC 598 2415 GCUGUGGAGACAGGUCCUC 399 2415 GCUGUGGAGACAGGUCCUC 399 2437 GAGGACCUGUCUCCACAGC 599 2433 CGCCCCCUACAACCCCCCU 400 2433 CGCCCCCUACAACCCCCCU 400 2455 AGGGGGGUUGUAGGGGGCG 600 2451 UGAGGGCCCCAGACCCCUC 401 2451 UGAGGGCCCCAGACCCCUC 401 2473 GAGGGGUCUGGGGCCCUCA 601 2469 CUCCAGGUGGGGUUCCACU 402 2469 CUCCAGGUGGGGUUCCACU 402 2491 AGUGGAACCCCACCUGGAG 602 2487 UGGAGGCCAGUCUGUGUCC 403 2487 UGGAGGCCAGUCUGUGUCC 403 2509 GGACACAGACUGGCCUCCA 603 2505 CGGCCUCCCUGGCACCCUC 404 2505 CGGCCUCCCUGGCACCCUC 404 2527 GAGGGUGCCAGGGAGGCCG 604 2523 CGGGCAUCUCAGAGAAGAG 405 2523 CGGGCAUCUCAGAGAAGAG 405 2545 CUCUUCUCUGAGAUGCCCG 605 2541 GUAAAUCCUCAUCAUCCUU 406 2541 GUAAAUCCUCAUCAUCCUU 406 2563 AAGGAUGAUGAGGAUUUAC 606 2559 UCCAUCCUGCCCCUGGCAA 407 2559 UCCAUCCUGCCCCUGGCAA 407 2581 UUGCCAGGGGCAGGAUGGA 607 2577 AUGCUCAGAGCUCAAGCCA 408 2577 AUGCUCAGAGCUCAAGCCA 408 2599 UGGCUUGAGCUCUGAGCAU 608 2595 AGACCCCCAAAAUCGUGAA 409 2595 AGACCCCCAAAAUCGUGAA 409 2617 UUCACGAUUUUGGGGGUCU 609 2613 ACUUUGUCUCCGUGGGACC 410 2613 ACUUUGUCUCCGUGGGACC 410 2635 GGUCCCACGGAGACAAAGU 610 2631 CCACAUACAUGAGGGUCUC 411 2631 CCACAUACAUGAGGGUCUC 411 2653 GAGACCCUCAUGUAUGUGG 611 2649 CUUAGGUGCAUGUCCUCUU 412 2649 CUUAGGUGCAUGUCCUCUU 412 2671 AAGAGGACAUGCACCUAAG 612 2667 UGUUGCUGAGUCUGCAGAU 413 2667 UGUUGCUGAGUCUGCAGAU 413 2689 AUCUGCAGACUCAGCAACA 613 2685 UGAGGACUAGGGCUUAUCC 414 2685 UGAGGACUAGGGCUUAUCC 414 2707 GGAUAAGCCCUAGUCCUCA 614 2703 CAUGCCUGGGAAAUGCCAC 415 2703 CAUGCCUGGGAAAUGCCAC 415 2725 GUGGCAUUUCCCAGGCAUG 615 2721 CCUCCUGGAAGGCAGCCAG 416 2721 CCUCCUGGAAGGCAGCCAG 416 2743 CUGGCUGCCUUCCAGGAGG 616 2739 GGCUGGCAGAUUUCCAAAA 417 2739 GGCUGGCAGAUUUCCAAAA 417 2761 UUUUGGAAAUCUGCCAGCC 617 2757 AGACUUGAAGAACCAUGGU 418 2757 AGACUUGAAGAACCAUGGU 418 2779 ACCAUGGUUCUUCAAGUCU 618 2775 UAUGAAGGUGAUUGGCCCC 419 2775 UAUGAAGGUGAUUGGCCCC 419 2797 GGGGCCAAUCACCUUCAUA 619 2793 CACUGACGUUGGCCUAACA 420 2793 CACUGACGUUGGCCUAACA 420 2815 UGUUAGGCCAACGUCAGUG 620 2811 ACUGGGCUGCAGAGACUGG 421 2811 ACUGGGCUGCAGAGACUGG 421 2833 CCAGUCUCUGCAGCCCAGU 621 2829 GACCCCGCCCAGCAUUGGG 422 2829 GACCCCGCCCAGCAUUGGG 422 2851 CCCAAUGCUGGGCGGGGUC 622 2847 GCUGGGCUCGCCACAUCCC 423 2847 GCUGGGCUCGCCACAUCCC 423 2869 GGGAUGUGGCGAGCCCAGC 623 2865 CAUGAGAGUAGAGGGCACU 424 2865 CAUGAGAGUAGAGGGCACU 424 2887 AGUGCCCUCUACUCUCAUG 624 2883 UGGGUCGCCGUGCCCCACG 425 2883 UGGGUCGCCGUGCCCCACG 425 2905 CGUGGGGCACGGCGACCCA 625 2901 GGCAGGCCCCUGCAGGAAA 426 2901 GGCAGGCCCCUGCAGGAAA 426 2923 UUUCCUGCAGGGGCCUGCC 626 2919 AACUGAGGCCCUUGGGCAC 427 2919 AACUGAGGCCCUUGGGCAC 427 2941 GUGCCCAAGGGCCUCAGUU 627 2937 CCUCGACUUGUGAACGAGU 428 2937 CCUCGACUUGUGAACGAGU 428 2959 ACUCGUUCACAAGUCGAGG 628 2955 UUGUUGGCUGCUCCCUCCA 429 2955 UUGUUGGCUGCUCCCUCCA 429 2977 UGGAGGGAGCAGCCAACAA 629 2973 ACAGCUUCUGCAGCAGACU 430 2973 ACAGCUUCUGCAGCAGACU 430 2995 AGUCUGCUGCAGAAGCUGU 630 2991 UGUCCCUGUUGUAACUGCC 431 2991 UGUCCCUGUUGUAACUGCC 431 3013 GGCAGUUACAACAGGGACA 631 3009 CCAAGGCAUGUUUUGCCCA 432 3009 CCAAGGCAUGUUUUGCCCA 432 3031 UGGGCAAAACAUGCCUUGG 632 3027 ACCAGAUCAUGGCCCACGU 433 3027 ACCAGAUCAUGGCCCACGU 433 3049 ACGUGGGCCAUGAUCUGGU 633 3045 UGGAGGCCCACCUGCCUCU 434 3045 UGGAGGCCCACCUGCCUCU 434 3067 AGAGGCAGGUGGGCCUCCA 634 3063 UGUCUCACUGAACUAGAAG 435 3063 UGUCUCACUGAACUAGAAG 435 3085 CUUCUAGUUCAGUGAGACA 635 3081 GCCGAGCCUAGAAACUAAC 436 3081 GCCGAGCCUAGAAACUAAC 436 3103 GUUAGUUUCUAGGCUCGGC 636 3099 CACAGCCAUCAAGGGAAUG 437 3099 CACAGCCAUCAAGGGAAUG 437 3121 CAUUCCCUUGAUGGCUGUG 637 3117 GACUUGGGCGGCCUUGGGA 438 3117 GACUUGGGCGGCCUUGGGA 438 3139 UCCCAAGGCCGCCCAAGUC 638 3135 AAAUCGAUGAGAAAUUGAA 439 3135 AAAUCGAUGAGAAAUUGAA 439 3157 UUCAAUUUCUCAUCGAUUU 639 3153 ACUUCAGGGAGGGUGGUCA 440 3153 ACUUCAGGGAGGGUGGUCA 440 3175 UGACCACCCUCCCUGAAGU 640 3171 AUUGCCUAGAGGUGCUCAU 441 3171 AUUGCCUAGAGGUGCUCAU 441 3193 AUGAGCACCUCUAGGCAAU 641 3189 UUCAUUUAACAGAGCUUCC 442 3189 UUCAUUUAACAGAGCUUCC 442 3211 GGAAGCUCUGUUAAAUGAA 642 3207 CUUAGGUUGAUGCUGGAGG 443 3207 CUUAGGUUGAUGCUGGAGG 443 3229 CCUCCAGCAUCAACCUAAG 643 3225 GCAGAAUCCCGGCUGUCAA 444 3225 GCAGAAUCCCGGCUGUCAA 444 3247 UUGACAGCCGGGAUUCUGC 644 3243 AGGGGUGUUCAGUUAAGGG 445 3243 AGGGGUGUUCAGUUAAGGG 445 3265 CCCUUAACUGAACACCCCU 645 3261 GGAGCAACAGAGGACAUGA 446 3261 GGAGCAACAGAGGACAUGA 446 3283 UCAUGUCCUCUGUUGCUCC 646 3279 AAAAAUUGCUAUGACUAAA 447 3279 AAAAAUUGCUAUGACUAAA 447 3301 UUUAGUCAUAGCAAUUUUU 647 3297 AGCAGGGACAAUUUGCUGC 448 3297 AGCAGGGACAAUUUGCUGC 448 3319 GCAGCAAAUUGUCCCUGCU 648 3315 CCAAACACCCAUGCCCAGC 449 3315 CCAAACACCCAUGCCCAGC 449 3337 GCUGGGCAUGGGUGUUUGG 649 3333 CUGUAUGGCUGGGGGCUCC 450 3333 CUGUAUGGCUGGGGGCUCC 450 3355 GGAGCCCCCAGCCAUACAG 650 3351 CUCGUAUGCAUGGAACCCC 451 3351 CUCGUAUGCAUGGAACCCC 451 3373 GGGGUUCCAUGCAUACGAG 651 3369 CCAGAAUAAAUAUGCUCAG 452 3369 CCAGAAUAAAUAUGCUCAG 452 3391 CUGAGCAUAUUUAUUCUGG 652 3387 GCCACCCUGUGGGCCGGGC 453 3387 GCCACCCUGUGGGCCGGGC 453 3409 GCCCGGCCCACAGGGUGGC 653 3405 CAAUCCAGACAGCAGGCAU 454 3405 CAAUCCAGACAGCAGGCAU 454 3427 AUGCCUGCUGUCUGGAUUG 654 3423 UAAGGCACCAGUUACCCUG 455 3423 UAAGGCACCAGUUACCCUG 455 3445 CAGGGUAACUGGUGCCUUA 655 3441 GCAUGUUGGCCCAGACCUC 456 3441 GCAUGUUGGCCCAGACCUC 456 3463 GAGGUCUGGGCCAACAUGC 656 3459 CAGGUGCUAGGGAAGGCGG 457 3459 CAGGUGCUAGGGAAGGCGG 457 3481 CCGCCUUCCCUAGCACCUG 657 3477 GGAACCUUGGGUUGAGUAA 458 3477 GGAACCUUGGGUUGAGUAA 458 3499 UUACUCAACCCAAGGUUCC 658 3495 AUGCUCGUCUGUGUGUUUU 459 3495 AUGCUCGUCUGUGUGUUUU 459 3517 AAAACACACAGACGAGCAU 659 3513 UAGUUUCAUCACCUGUUAU 460 3513 UAGUUUCAUCACCUGUUAU 460 3535 AUAACAGGUGAUGAAACUA 660 3531 UCUGUGUUUGCUGAGGAGA 461 3531 UCUGUGUUUGCUGAGGAGA 461 3553 UCUCCUCAGCAAACACAGA 661 3549 AGUGGAACAGAAGGGGUGG 462 3549 AGUGGAACAGAAGGGGUGG 462 3571 CCACCCCUUCUGUUCCACU 662 3567 GAGUUUUGUAUAAAUAAAG 463 3567 GAGUUUUGUAUAAAUAAAG 463 3589 CUUUAUUUAUACAAAACUC 663 3577 UAAAUAAAGUUUCUUUGUC 464 3577 UAAAUAAAGUUUCUUUGUC 464 3599 GACAAAGAAACUUUAUUUA 664 IL13 NM_002188 3 GCCACCCAGCCUAUGCAUC 665 3 GCCACCCAGCCUAUGCAUC 665 25 GAUGCAUAGGCUGGGUGGC 736 21 CCGCUCCUCAAUCCUCUCC 666 21 CCGCUCCUCAAUCCUCUCC 666 43 GGAGAGGAUUGAGGAGCGG 737 39 CUGUUGGCACUGGGCCUCA 667 39 CUGUUGGCACUGGGCCUCA 667 61 UGAGGCCCAGUGCCAACAG 738 57 AUGGCGCUUUUGUUGACCA 668 57 AUGGCGCUUUUGUUGACCA 668 79 UGGUCAACAAAAGCGCCAU 739 75 ACGGUCAUUGCUCUCACUU 669 75 ACGGUCAUUGCUCUCACUU 669 97 AAGUGAGAGCAAUGACCGU 740 93 UGCCUUGGCGGCUUUGCCU 670 93 UGCCUUGGCGGCUUUGCCU 670 115 AGGCAAAGCCGCCAAGGCA 741 111 UCCCCAGGCCCUGUGCCUC 671 111 UCCCCAGGCCCUGUGCCUC 671 133 GAGGCACAGGGCCUGGGGA 742 129 CCCUCUACAGCCCUCAGGG 672 129 CCCUCUACAGCCCUCAGGG 672 151 CCCUGAGGGCUGUAGAGGG 743 147 GAGCUCAUUGAGGAGCUGG 673 147 GAGCUCAUUGAGGAGCUGG 673 169 CCAGCUCCUCAAUGAGCUC 744 165 GUCAACAUCACCCAGAACC 674 165 GUCAACAUCACCCAGAACC 674 187 GGUUCUGGGUGAUGUUGAC 745 183 CAGAAGGCUCCGCUCUGCA 675 183 CAGAAGGCUCCGCUCUGCA 675 205 UGCAGAGCGGAGCCUUCUG 746 201 AAUGGCAGCAUGGUAUGGA 676 201 AAUGGCAGCAUGGUAUGGA 676 223 UCCAUACCAUGCUGCCAUU 747 219 AGCAUCAACCUGACAGCUG 677 219 AGCAUCAACCUGACAGCUG 677 241 CAGCUGUCAGGUUGAUGCU 748 237 GGCAUGUACUGUGCAGCCC 678 237 GGCAUGUACUGUGCAGCCC 678 259 GGGCUGCACAGUACAUGCC 749 255 CUGGAAUCCCUGAUCAACG 679 255 CUGGAAUCCCUGAUCAACG 679 277 CGUUGAUCAGGGAUUCCAG 750 273 GUGUCAGGCUGCAGUGCCA 680 273 GUGUCAGGCUGCAGUGCCA 680 295 UGGCACUGCAGCCUGACAC 751 291 AUCGAGAAGACCCAGAGGA 681 291 AUCGAGAAGACCCAGAGGA 681 313 UCCUCUGGGUCUUCUCGAU 752 309 AUGCUGAGCGGAUUCUGCC 682 309 AUGCUGAGCGGAUUCUGCC 682 331 GGCAGAAUCCGCUCAGCAU 753 327 CCGCACAAGGUCUCAGCUG 683 327 CCGCACAAGGUCUCAGCUG 683 349 CAGCUGAGACCUUGUGCGG 754 345 GGGCAGUUUUCCAGCUUGC 684 345 GGGCAGUUUUCCAGCUUGC 684 367 GCAAGCUGGAAAACUGCCC 755 363 CAUGUCCGAGACACCAAAA 685 363 CAUGUCCGAGACACCAAAA 685 385 UUUUGGUGUCUCGGACAUG 756 381 AUCGAGGUGGCCCAGUUUG 686 381 AUCGAGGUGGCCCAGUUUG 686 403 CAAACUGGGCCACCUCGAU 757 399 GUAAAGGACCUGCUCUUAC 687 399 GUAAAGGACCUGCUCUUAC 687 421 GUAAGAGCAGGUCCUUUAC 758 417 CAUUUAAAGAAACUUUUUC 688 417 CAUUUAAAGAAACUUUUUC 688 439 GAAAAAGUUUCUUUAAAUG 759 435 CGCGAGGGACAGUUCAACU 689 435 CGCGAGGGACAGUUCAACU 689 457 AGUUGAACUGUCCCUCGCG 760 453 UGAAACUUCGAAAGCAUCA 690 453 UGAAACUUCGAAAGCAUCA 690 475 UGAUGCUUUCGAAGUUUCA 761 471 AUUAUUUGCAGAGACAGGA 691 471 AUUAUUUGCAGAGACAGGA 691 493 UCCUGUCUCUGCAAAUAAU 762 489 ACCUGACUAUUGAAGUUGC 692 489 ACCUGACUAUUGAAGUUGC 692 511 GCAACUUCAAUAGUCAGGU 763 507 CAGAUUCAUUUUUCUUUCU 693 507 CAGAUUCAUUUUUCUUUCU 693 529 AGAAAGAAAAAUGAAUCUG 764 525 UGAUGUCAAAAAUGUCUUG 694 525 UGAUGUCAAAAAUGUCUUG 694 547 CAAGACAUUUUUGACAUCA 765 543 GGGUAGGCGGGAAGGAGGG 695 543 GGGUAGGCGGGAAGGAGGG 695 565 CCCUCCUUCCCGCCUACCC 766 561 GUUAGGGAGGGGUAAAAUU 696 561 GUUAGGGAGGGGUAAAAUU 696 583 AAUUUUACCCCUCCCUAAC 767 579 UCCUUAGCUUAGACCUCAG 697 579 UCCUUAGCUUAGACCUCAG 697 601 CUGAGGUCUAAGCUAAGGA 768 597 GCCUGUGCUGCCCGUCUUC 698 597 GCCUGUGCUGCCCGUCUUC 698 619 GAAGACGGGCAGCACAGGC 769 615 CAGCCUAGCCGACCUCAGC 699 615 CAGCCUAGCCGACCUCAGC 699 637 GCUGAGGUCGGCUAGGCUG 770 633 CCUUCCCCUUGCCCAGGGC 700 633 CCUUCCCCUUGCCCAGGGC 700 655 GCCCUGGGCAAGGGGAAGG 771 651 CUCAGCCUGGUGGGCCUCC 701 651 CUCAGCCUGGUGGGCCUCC 701 673 GGAGGCCCACCAGGCUGAG 772 669 CUCUGUCCAGGGCCCUGAG 702 669 CUCUGUCCAGGGCCCUGAG 702 691 CUCAGGGCCCUGGACAGAG 773 687 GCUCGGUGGACCCAGGGAU 703 687 GCUCGGUGGACCCAGGGAU 703 709 AUCCCUGGGUCCACCGAGC 774 705 UGACAUGUCCCUACACCCC 704 705 UGACAUGUCCCUACACCCC 704 727 GGGGUGUAGGGACAUGUCA 775 723 CUCCCCUGCCCUAGAGCAC 705 723 CUCCCCUGCCCUAGAGCAC 705 745 GUGCUCUAGGGCAGGGGAG 776 741 CACUGUAGCAUUACAGUGG 706 741 CACUGUAGCAUUACAGUGG 706 763 CCACUGUAAUGCUACAGUG 777 759 GGUGCCCCCCUUGCCAGAC 707 759 GGUGCCCCCCUUGCCAGAC 707 781 GUCUGGCAAGGGGGGCACC 778 777 CAUGUGGUGGGACAGGGAC 708 777 CAUGUGGUGGGACAGGGAC 708 799 GUCCCUGUCCCACCACAUG 779 795 CCCACUUCACACACAGGCA 709 795 CCCACUUCACACACAGGCA 709 817 UGCCUGUGUGUGAAGUGGG 780 813 AACUGAGGCAGACAGCAGC 710 813 AACUGAGGCAGACAGCAGC 710 835 GCUGCUGUCUGCCUCAGUU 781 831 CUCAGGCACACUUCUUCUU 711 831 CUCAGGCACACUUCUUCUU 711 853 AAGAAGAAGUGUGCCUGAG 782 849 UGGUCUUAUUUAUUAUUGU 712 849 UGGUCUUAUUUAUUAUUGU 712 871 ACAAUAAUAAAUAAGACCA 783 867 UGUGUUAUUUAAAUGAGUG 713 867 UGUGUUAUUUAAAUGAGUG 713 889 CACUCAUUUAAAUAACACA 784 885 GUGUUUGUCACCGUUGGGG 714 885 GUGUUUGUCACCGUUGGGG 714 907 CCCCAACGGUGACAAACAC 785 903 GAUUGGGGAAGACUGUGGC 715 903 GAUUGGGGAAGACUGUGGC 715 925 GCCACAGUCUUCCCCAAUC 786 921 CUGCUAGCACUUGGAGCCA 716 921 CUGCUAGCACUUGGAGCCA 716 943 UGGCUCCAAGUGCUAGCAG 787 939 AAGGGUUCAGAGACUCAGG 717 939 AAGGGUUCAGAGACUCAGG 717 961 CCUGAGUCUCUGAACCCUU 788 957 GGCCCCAGCACUAAAGCAG 718 957 GGCCCCAGCACUAAAGCAG 718 979 CUGCUUUAGUGCUGGGGCC 789 975 GUGGACACCAGGAGUCCCU 719 975 GUGGACACCAGGAGUCCCU 719 997 AGGGACUCCUGGUGUCCAC 790 933 UGGUAAUAAGUACUGUGUA 720 993 UGGUAAUAAGUACUGUGUA 720 1015 UACACAGUACUUAUUACCA 791 1011 ACAGAAUUCUGCUACCUCA 721 1011 ACAGAAUUCUGCUACCUCA 721 1033 UGAGGUAGCAGAAUUCUGU 792 1029 ACUGGGGUCCUGGGGCCUC 722 1029 ACUGGGGUCCUGGGGCCUC 722 1051 GAGGCCCCAGGACCCCAGU 793 1047 CGGAGCCUCAUCCGAGGCA 723 1047 CGGAGCCUCAUCCGAGGCA 723 1069 UGCCUCGGAUGAGGCUCCG 794 1065 AGGGUCAGGAGAGGGGCAG 724 1065 AGGGUCAGGAGAGGGGCAG 724 1087 CUGCCCCUCUCCUGACCCU 795 1083 GAACAGCCGCUCCUGUCUG 725 1083 GAACAGCCGCUCCUGUCUG 725 1105 CAGACAGGAGCGGCUGUUC 796 1101 GCCAGCCAGCAGCCAGCUC 726 1101 GCCAGCCAGCAGCCAGCUC 726 1123 GAGCUGGCUGCUGGCUGGC 797 1119 CUCAGCCAACGAGUAAUUU 727 1119 CUCAGCCAACGAGUAAUUU 727 1141 AAAUUACUCGUUGGCUGAG 798 1137 UAUUGUUUUUCCUUGUAUU 728 1137 UAUUGUUUUUCCUUGUAUU 728 1159 AAUACAAGGAAAAACAAUA 799 1155 UUAAAUAUUAAAUAUGUUA 729 1155 UUAAAUAUUAAAUAUGUUA 729 1177 UAACAUAUUUAAUAUUUAA 800 1173 AGCAAAGAGUUAAUAUAUA 730 1173 AGCAAAGAGUUAAUAUAUA 730 1195 UAUAUAUUAACUCUUUGCU 801 1191 AGAAGGGUACCUUGAACAC 731 1191 AGAAGGGUACCUUGAACAC 731 1213 GUGUUCAAGGUACCCUUCU 802 1209 CUGGGGGAGGGGACAUUGA 732 1209 CUGGGGGAGGGGACAUUGA 732 1231 UCAAUGUCCCCUCCCCCAG 803 1227 AACAAGUUGUUUCAUUGAC 733 1227 AACAAGUUGUUUCAUUGAC 733 1249 GUCAAUGAAACAACUUGUU 804 1245 CUAUCAAACUGAAGCCAGA 734 1245 CUAUCAAACUGAAGCCAGA 734 1267 UCUGGCUUCAGUUUGAUAG 805 1262 GAAAUAAAGUUGGUGACAG 735 1262 GAAAUAAAGUUGGUGACAG 735 1284 CUGUCACCAACUUUAUUUC 806 IL 13RA1 NM_001560 3 CCAAGGCUCCAGCCCGGCC 807 3 CCAAGGCUCCAGCCCGGCC 807 25 GGCCGGGCUGGAGCCUUGG 1030 21 CGGGCUCCGAGGCGAGAGG 808 21 CGGGCUCCGAGGCGAGAGG 808 43 CCUCUCGCCUCGGAGCCCG 1031 39 GCUGCAUGGAGUGGCCGGC 809 39 GCUGCAUGGAGUGGCCGGC 809 61 GCCGGCCACUCCAUGCAGC 1032 57 CGCGGCUCUGCGGGCUGUG 810 57 CGCGGCUCUGCGGGCUGUG 810 79 CACAGCCCGCAGAGCCGCG 1033 75 GGGCGCUGCUGCUCUGCGC 811 75 GGGCGCUGCUGCUCUGCGC 811 97 GCGCAGAGCAGCAGCGCCC 1034 93 CCGGCGGCGGGGGCGGGGG 812 93 CCGGCGGCGGGGGCGGGGG 812 115 CCCCCGCCCCCGCCGCCGG 1035 111 GCGGGGGCGCCGCGCCUAC 813 111 GCGGGGGCGCCGCGCCUAC 813 133 GUAGGCGCGGCGCCCCCGC 1036 129 CGGAAACUCAGCCACCUGU 814 129 CGGAAACUCAGCCACCUGU 814 151 ACAGGUGGCUGAGUUUCCG 1037 147 UGACAAAUUUGAGUGUCUC 815 147 UGACAAAUUUGAGUGUCUC 815 169 GAGACACUCAAAUUUGUCA 1038 165 CUGUUGAAAACCUCUGCAC 816 165 CUGUUGAAAACCUCUGCAC 816 187 GUGCAGAGGUUUUCAACAG 1039 183 CAGUAAUAUGGACAUGGAA 817 183 CAGUAAUAUGGACAUGGAA 817 205 UUCCAUGUCCAUAUUACUG 1040 201 AUCCACCCGAGGGAGCCAG 818 201 AUCCACCCGAGGGAGCCAG 818 223 CUGGCUCCCUCGGGUGGAU 1041 219 GCUCAAAUUGUAGUCUAUG 819 219 GCUCAAAUUGUAGUCUAUG 819 241 CAUAGACUACAAUUUGAGC 1042 237 GGUAUUUUAGUCAUUUUGG 820 237 GGUAUUUUAGUCAUUUUGG 820 259 CCAAAAUGACUAAAAUACC 1043 255 GCGACAAACAAGAUAAGAA 821 255 GCGACAAACAAGAUAAGAA 821 277 UUCUUAUCUUGUUUGUCGC 1044 273 AAAUAGCUCCGGAAACUCG 822 273 AAAUAGCUCCGGAAACUCG 822 295 CGAGUUUCCGGAGCUAUUU 1045 291 GUCGUUCAAUAGAAGUACC 823 291 GUCGUUCAAUAGAAGUACC 823 313 GGUACUUCUAUUGAACGAC 1046 309 CCCUGAAUGAGAGGAUUUG 824 309 CCCUGAAUGAGAGGAUUUG 824 331 CAAAUCCUCUCAUUCAGGG 1047 327 GUCUGCAAGUGGGGUCCCA 825 327 GUCUGCAAGUGGGGUCCCA 825 349 UGGGACCCCACUUGCAGAC 1048 345 AGUGUAGCACCAAUGAGAG 826 345 AGUGUAGCACCAAUGAGAG 826 367 CUCUCAUUGGUGCUACACU 1049 363 GUGAGAAGCCUAGCAUUUU 827 363 GUGAGAAGCCUAGCAUUUU 827 385 AAAAUGCUAGGCUUCUCAC 1050 381 UGGUUGAAAAAUGCAUCUC 828 381 UGGUUGAAAAAUGCAUCUC 828 403 GAGAUGCAUUUUUCAACCA 1051 399 CACCCCCAGAAGGUGAUCC 829 399 CACCCCCAGAAGGUGAUCC 829 421 GGAUCACCUUCUGGGGGUG 1052 417 CUGAGUCUGCUGUGACUGA 830 417 CUGAGUCUGCUGUGACUGA 830 439 UCAGUCACAGCAGACUCAG 1053 435 AGCUUCAAUGCAUUUGGCA 831 435 AGCUUCAAUGCAUUUGGCA 831 457 UGCCAAAUGCAUUGAAGCU 1054 453 ACAACCUGAGCUACAUGAA 832 453 ACAACCUGAGCUACAUGAA 832 475 UUCAUGUAGCUCAGGUUGU 1055 471 AGUGUUCUUGGCUCCCUGG 833 471 AGUGUUCUUGGCUCCCUGG 833 493 CCAGGGAGCCAAGAACACU 1056 489 GAAGGAAUACCAGUCCCGA 834 489 GAAGGAAUACCAGUCCCGA 834 511 UCGGGACUGGUAUUCCUUC 1057 507 ACACUAACUAUACUCUCUA 835 507 ACACUAACUAUACUCUCUA 835 529 UAGAGAGUAUAGUUAGUGU 1058 525 ACUAUUGGCACAGAAGCCU 836 525 ACUAUUGGCACAGAAGCCU 836 547 AGGCUUCUGUGCCAAUAGU 1059 543 UGGAAAAAAUUCAUCAAUG 837 543 UGGAAAAAAUUCAUCAAUG 837 565 CAUUGAUGAAUUUUUUCCA 1060 561 GUGAAAACAUCUUUAGAGA 838 561 GUGAAAACAUCUUUAGAGA 838 583 UCUCUAAAGAUGUUUUCAC 1061 579 AAGGCCAAUACUUUGGUUG 839 579 AAGGCCAAUACUUUGGUUG 839 601 CAACCAAAGUAUUGGCCUU 1062 597 GUUCCUUUGAUCUGACCAA 840 597 GUUCCUUUGAUCUGACCAA 840 619 UUGGUCAGAUCAAAGGAAC 1063 615 AAGUGAAGGAUUCCAGUUU 841 615 AAGUGAAGGAUUCCAGUUU 841 637 AAACUGGAAUCCUUCACUU 1064 633 UUGAACAACACAGUGUCCA 842 633 UUGAACAACACAGUGUCCA 842 655 UGGACACUGUGUUGUUCAA 1065 651 AAAUAAUGGUCAAGGAUAA 843 651 AAAUAAUGGUCAAGGAUAA 843 673 UUAUCCUUGACCAUUAUUU 1066 669 AUGCAGGAAAAAUUAAACC 844 669 AUGCAGGAAAAAUUAAACC 844 691 GGUUUAAUUUUUCCUGCAU 1067 687 CAUCCUUCAAUAUAGUGCC 845 687 CAUCCUUCAAUAUAGUGCC 845 709 GGCACUAUAUUGAAGGAUG 1068 705 CUUUAACUUCCCGUGUGAA 846 705 CUUUAACUUCCCGUGUGAA 846 727 UUCACACGGGAAGUUAAAG 1069 723 AACCUGAUCCUCCACAUAU 847 723 AACCUGAUCCUCCACAUAU 847 745 AUAUGUGGAGGAUCAGGUU 1070 741 UUAAAAACCUCUCCUUCCA 848 741 UUAAAAACCUCUCCUUCCA 848 763 UGGAAGGAGAGGUUUUUAA 1071 759 ACAAUGAUGACCUAUAUGU 849 759 ACAAUGAUGACCUAUAUGU 849 781 ACAUAUAGGUCAUCAUUGU 1072 777 UGCAAUGGGAGAAUCCACA 850 777 UGCAAUGGGAGAAUCCACA 850 799 UGUGGAUUCUCCCAUUGCA 1073 795 AGAAUUUUAUUAGCAGAUG 851 795 AGAAUUUUAUUAGCAGAUG 851 817 CAUCUGCUAAUAAAAUUCU 1074 813 GCCUAUUUUAUGAAGUAGA 852 813 GCCUAUUUUAUGAAGUAGA 852 835 UCUACUUCAUAAAAUAGGC 1075 831 AAGUCAAUAACAGCCAAAC 853 831 AAGUCAAUAACAGCCAAAC 853 853 GUUUGGCUGUUAUUGACUU 1076 849 CUGAGACACAUAAUGUUUU 854 849 CUGAGACACAUAAUGUUUU 854 871 AAAACAUUAUGUGUCUCAG 1077 867 UCUACGUCCAAGAGGCUAA 855 867 UCUACGUCCAAGAGGCUAA 855 889 UUAGCCUCUUGGACGUAGA 1078 885 AAUGUGAGAAUCCAGAAUU 856 885 AAUGUGAGAAUCCAGAAUU 856 907 AAUUCUGGAUUCUCACAUU 1079 903 UUGAGAGAAAUGUGGAGAA 857 903 UUGAGAGAAAUGUGGAGAA 857 925 UUCUCCACAUUUCUCUCAA 1080 921 AUACAUCUUGUUUCAUGGU 858 921 AUACAUCUUGUUUCAUGGU 858 943 ACCAUGAAACAAGAUGUAU 1081 939 UCCCUGGUGUUCUUCCUGA 859 939 UCCCUGGUGUUCUUCCUGA 859 961 UCAGGAAGAACACCAGGGA 1082 957 AUACUUUGAACACAGUCAG 860 957 AUACUUUGAACACAGUCAG 860 979 CUGACUGUGUUCAAAGUAU 1083 975 GAAUAAGAGUCAAAACAAA 861 975 GAAUAAGAGUCAAAACAAA 861 997 UUUGUUUUGACUCUUAUUC 1084 993 AUAAGUUAUGCUAUGAGGA 862 993 AUAAGUUAUGCUAUGAGGA 862 1015 UCCUCAUAGCAUAACUUAU 1085 1011 AUGACAAACUCUGGAGUAA 863 1011 AUGACAAACUCUGGAGUAA 863 1033 UUACUCCAGAGUUUGUCAU 1086 1029 AUUGGAGCCAAGAAAUGAG 864 1029 AUUGGAGCCAAGAAAUGAG 864 1051 CUCAUUUCUUGGCUCCAAU 1087 1047 GUAUAGGUAAGAAGCGCAA 865 1047 GUAUAGGUAAGAAGCGCAA 865 1069 UUGCGCUUCUUACCUAUAC 1088 1065 AUUCCACACUCUACAUAAC 866 1065 AUUCCACACUCUACAUAAC 866 1087 GUUAUGUAGAGUGUGGAAU 1089 1083 CCAUGUUACUCAUUGUUCC 867 1083 CCAUGUUACUCAUUGUUCC 867 1105 GGAACAAUGAGUAACAUGG 1090 1101 CAGUCAUCGUCGCAGGUGC 868 1101 CAGUCAUCGUCGCAGGUGC 868 1123 GCACCUGCGACGAUGACUG 1091 1119 CAAUCAUAGUACUCCUGCU 869 1119 CAAUCAUAGUACUCCUGCU 869 1141 AGCAGGAGUACUAUGAUUG 1092 1137 UUUACCUAAAAAGGCUCAA 870 1137 UUUACCUAAAAAGGCUCAA 870 1159 UUGAGCCUUUUUAGGUAAA 1093 1155 AGAUUAUUAUAUUCCCUCC 871 1155 AGAUUAUUAUAUUCCCUCC 871 1177 GGAGGGAAUAUAAUAAUCU 1094 1173 CAAUUCCUGAUCCUGGCAA 872 1173 CAAUUCCUGAUCCUGGCAA 872 1195 UUGCCAGGAUCAGGAAUUG 1095 1191 AGAUUUUUAAAGAAAUGUU 873 1191 AGAUUUUUAAAGAAAUGUU 873 1213 AACAUUUCUUUAAAAAUCU 1096 1209 UUGGAGACCAGAAUGAUGA 874 1209 UUGGAGACCAGAAUGAUGA 874 1231 UCAUCAUUCUGGUCUCCAA 1097 1227 AUACUCUGCACUGGAAGAA 875 1227 AUACUCUGCACUGGAAGAA 875 1249 UUCUUCCAGUGCAGAGUAU 1098 1245 AGUACGACAUCUAUGAGAA 876 1245 AGUACGACAUCUAUGAGAA 876 1267 UUCUCAUAGAUGUCGUACU 1099 1263 AGCAAACCAAGGAGGAAAC 877 1263 AGCAAACCAAGGAGGAAAC 877 1285 GUUUCCUCCUUGGUUUGCU 1100 1281 CCGACUCUGUAGUGCUGAU 878 1281 CCGACUCUGUAGUGCUGAU 878 1303 AUCAGCACUACAGAGUCGG 1101 1299 UAGAAAACCUGAAGAAAGC 879 1299 UAGAAAACCUGAAGAAAGC 879 1321 GCUUUCUUCAGGUUUUCUA 1102 1317 CCUCUCAGUGAUGGAGAUA 880 1317 CCUCUCAGUGAUGGAGAUA 880 1339 UAUCUCCAUCACUGAGAGG 1103 1335 AAUUUAUUUUUACCUUCAC 881 1335 AAUUUAUUUUUACCUUCAC 881 1357 GUGAAGGUAAAAAUAAAUU 1104 1353 CUGUGACCUUGAGAAGAUU 882 1353 CUGUGACCUUGAGAAGAUU 882 1375 AAUCUUCUCAAGGUCACAG 1105 1371 UCUUCCCAUUCUCCAUUUG 883 1371 UCUUCCCAUUCUCCAUUUG 883 1393 CAAAUGGAGAAUGGGAAGA 1106 1389 GUUAUCUGGGAACUUAUUA 884 1389 GUUAUCUGGGAACUUAUUA 884 1411 UAAUAAGUUCCCAGAUAAC 1107 1407 AAAUGGAAACUGAAACUAC 885 1407 AAAUGGAAACUGAAACUAC 885 1429 GUAGUUUCAGUUUCCAUUU 1108 1425 CUGCACCAUUUAAAAACAG 886 1425 CUGCACCAUUUAAAAACAG 886 1447 CUGUUUUUAAAUGGUGCAG 1109 1443 GGCAGCUCAUAAGAGCCAC 887 1443 GGCAGCUCAUAAGAGCCAC 887 1465 GUGGCUCUUAUGAGCUGCC 1110 1461 CAGGUCUUUAUGUUGAGUC 888 1461 CAGGUCUUUAUGUUGAGUC 888 1483 GACUCAACAUAAAGACCUG 1111 1479 CGCGCACCGAAAAACUAAA 889 1479 CGCGCACCGAAAAACUAAA 889 1501 UUUAGUUUUUCGGUGCGCG 1112 1497 AAAUAAUGGGCGCUUUGGA 890 1497 AAAUAAUGGGCGCUUUGGA 890 1519 UCCAAAGCGCCCAUUAUUU 1113 1515 AGAAGAGUGUGGAGUCAUU 891 1515 AGAAGAGUGUGGAGUCAUU 891 1537 AAUGACUCCACACUCUUCU 1114 1533 UCUCAUUGAAUUAUAAAAG 892 1533 UCUCAUUGAAUUAUAAAAG 892 1555 CUUUUAUAAUUCAAUGAGA 1115 1551 GCCAGCAGGCUUCAAACUA 893 1551 GCCAGCAGGCUUCAAACUA 893 1573 UAGUUUGAAGCCUGCUGGC 1116 1569 AGGGGACAAAGCAAAAAGU 894 1569 AGGGGACAAAGCAAAAAGU 894 1591 ACUUUUUGCUUUGUCCCCU 1117 1587 UGAUGAUAGUGGUGGAGUU 895 1587 UGAUGAUAGUGGUGGAGUU 895 1609 AACUCCACCACUAUCAUCA 1118 1605 UAAUCUUAUCAAGAGUUGU 896 1605 UAAUCUUAUCAAGAGUUGU 896 1627 ACAACUCUUGAUAAGAUUA 1119 1623 UGACAACUUCCUGAGGGAU 897 1623 UGACAACUUCCUGAGGGAU 897 1645 AUCCCUCAGGAAGUUGUCA 1120 1641 UCUAUACUUGCUUUGUGUU 898 1641 UCUAUACUUGCUUUGUGUU 898 1663 AACACAAAGCAAGUAUAGA 1121 1659 UCUUUGUGUCAACAUGAAC 899 1659 UCUUUGUGUCAACAUGAAC 899 1681 GUUCAUGUUGACACAAAGA 1122 1677 CAAAUUUUAUUUGUAGGGG 900 1677 CAAAUUUUAUUUGUAGGGG 900 1699 CCCCUACAAAUAAAAUUUG 1123 1695 GAACUCAUUUGGGGUGCAA 901 1695 GAACUCAUUUGGGGUGCAA 901 1717 UUGCACCCCAAAUGAGUUC 1124 1713 AAUGCUAAUGUCAAACUUG 902 1713 AAUGCUAAUGUCAAACUUG 902 1735 CAAGUUUGACAUUAGCAUU 1125 1731 GAGUCACAAAGAACAUGUA 903 1731 GAGUCACAAAGAACAUGUA 903 1753 UACAUGUUCUUUGUGACUC 1126 1749 AGAAAACAAAAUGGAUAAA 904 1749 AGAAAACAAAAUGGAUAAA 904 1771 UUUAUCCAUUUUGUUUUCU 1127 1767 AAUCUGAUAUGUAUUGUUU 905 1767 AAUCUGAUAUGUAUUGUUU 905 1789 AAACAAUACAUAUCAGAUU 1128 1785 UGGGAUCCUAUUGAACCAU 906 1785 UGGGAUCCUAUUGAACCAU 906 1807 AUGGUUCAAUAGGAUCCCA 1129 1803 UGUUUGUGGCUAUUAAAAC 907 1803 UGUUUGUGGCUAUUAAAAC 907 1825 GUUUUAAUAGCCACAAACA 1130 1821 CUCUUUUAACAGUCUGGGC 908 1821 CUCUUUUAACAGUCUGGGC 908 1843 GCCCAGACUGUUAAAAGAG 1131 1839 CUGGGUCCGGUGGCUCACG 909 1839 CUGGGUCCGGUGGCUCACG 909 1861 CGUGAGCCACCGGACCCAG 1132 1857 GCCUGUAAUCCCAGCAAUU 910 1857 GCCUGUAAUCCCAGCAAUU 910 1879 AAUUGCUGGGAUUACAGGC 1133 1875 UUGGGAGUCCGAGGCGGGC 911 1875 UUGGGAGUCCGAGGCGGGC 911 1897 GCCCGCCUCGGACUCCCAA 1134 1893 CGGAUCACUCGAGGUCAGG 912 1893 CGGAUCACUCGAGGUCAGG 912 1915 CCUGACCUCGAGUGAUCCG 1135 1911 GAGUUCCAGACCAGCCUGA 913 1911 GAGUUCCAGACCAGCCUGA 913 1933 UCAGGCUGGUCUGGAACUC 1136 1929 ACCAAAAUGGUGAAACCUC 914 1929 ACCAAAAUGGUGAAACCUC 914 1951 GAGGUUUCACCAUUUUGGU 1137 1947 CCUCUCUACUAAAACUACA 915 1947 CCUCUCUACUAAAACUACA 915 1969 UGUAGUUUUAGUAGAGAGG 1138 1965 AAAAAUUAACUGGGUGUGG 916 1965 AAAAAUUAACUGGGUGUGG 916 1987 CCACACCCAGUUAAUUUUU 1139 1983 GUGGCGCGUGCCUGUAAUC 917 1983 GUGGCGCGUGCCUGUAAUC 917 2005 GAUUACAGGCACGCGCCAC 1140 2001 CCCAGCUACUCGGGAAGCU 918 2001 CCCAGCUACUCGGGAAGCU 918 2023 AGCUUCCCGAGUAGCUGGG 1141 2019 UGAGGCAGGUGAAUUGUUU 919 2019 UGAGGCAGGUGAAUUGUUU 919 2041 AAACAAUUCACCUGCCUCA 1142 2037 UGAACCUGGGAGGUGGAGG 920 2037 UGAACCUGGGAGGUGGAGG 920 2059 CCUCCACCUCCCAGGUUCA 1143 2055 GUUGCAGUGAGCAGAGAUC 921 2055 GUUGCAGUGAGCAGAGAUC 921 2077 GAUCUCUGCUCACUGCAAC 1144 2073 CACACCACUGCACUCUAGC 922 2073 CACACCACUGCACUCUAGC 922 2095 GCUAGAGUGCAGUGGUGUG 1145 2091 CCUGGGUGACAGAGCAAGA 923 2091 CCUGGGUGACAGAGCAAGA 923 2113 UCUUGCUCUGUCACCCAGG 1146 2109 ACUCUGUCUAAAAAACAAA 924 2109 ACUCUGUCUAAAAAACAAA 924 2131 UUUGUUUUUUAGACAGAGU 1147 2127 AACAAAACAAAACAAAACA 925 2127 AACAAAACAAAACAAAACA 925 2149 UGUUUUGUUUUGUUUUGUU 1148 2145 AAAAAAACCUCUUAAUAUU 926 2145 AAAAAAACCUCUUAAUAUU 926 2167 AAUAUUAAGAGGUUUUUUU 1149 2163 UCUGGAGUCAUCAUUCCCU 927 2163 UCUGGAGUCAUCAUUCCCU 927 2185 AGGGAAUGAUGACUCCAGA 1150 2181 UUCGACAGCAUUUUCCUCU 928 2181 UUCGACAGCAUUUUCCUCU 928 2203 AGAGGAAAAUGCUGUCGAA 1151 2199 UGCUUUGAAAGCCCCAGAA 929 2199 UGCUUUGAAAGCCCCAGAA 929 2221 UUCUGGGGCUUUCAAAGCA 1152 2217 AAUCAGUGUUGGCCAUGAU 930 2217 AAUCAGUGUUGGCCAUGAU 930 2239 AUCAUGGCCAACACUGAUU 1153 2235 UGACAACUACAGAAAAACC 931 2235 UGACAACUACAGAAAAACC 931 2257 GGUUUUUCUGUAGUUGUCA 1154 2253 CAGAGGCAGCUUCUUUGCC 932 2253 CAGAGGCAGCUUCUUUGCC 932 2275 GGCAAAGAAGCUGCCUCUG 1155 2271 CAAGACCUUUCAAAGCCAU 933 2271 CAAGACCUUUCAAAGCCAU 933 2293 AUGGCUUUGAAAGGUCUUG 1156 2289 UUUUAGGCUGUUAGGGGCA 934 2289 UUUUAGGCUGUUAGGGGCA 934 2311 UGCCCCUAACAGCCUAAAA 1157 2307 AGUGGAGGUAGAAUGACUC 935 2307 AGUGGAGGUAGAAUGACUC 935 2329 GAGUCAUUCUACCUCCACU 1158 2325 CCUUGGGUAUUAGAGUUUC 936 2325 CCUUGGGUAUUAGAGUUUC 936 2347 GAAACUCUAAUACCCAAGG 1159 2343 CAACCAUGAAGUCUCUAAC 937 2343 CAACCAUGAAGUCUCUAAC 937 2365 GUUAGAGACUUCAUGGUUG 1160 2361 CAAUGUAUUUUCUUCACCU 938 2361 CAAUGUAUUUUCUUCACCU 938 2383 AGGUGAAGAAAAUACAUUG 1161 2379 UCUGCUACUCAAGUAGCAU 939 2379 UCUGCUACUCAAGUAGCAU 939 2401 AUGCUACUUGAGUAGCAGA 1162 2397 UUUACUGUGUCUUUGGUUU 940 2397 UUUACUGUGUCUUUGGUUU 940 2419 AAACCAAAGACACAGUAAA 1163 2415 UGUGCUAGGCCCCCGGGUG 941 2415 UGUGCUAGGCCCCCGGGUG 941 2437 CACCCGGGGGCCUAGCACA 1164 2433 GUGAAGCACAGACCCCUUC 942 2433 GUGAAGCACAGACCCCUUC 942 2455 GAAGGGGUCUGUGCUUCAC 1165 2451 CCAGGGGUUUACAGUCUAU 943 2451 CCAGGGGUUUACAGUCUAU 943 2473 AUAGACUGUAAACCCCUGG 1166 2469 UUUGAGACUCCUCAGUUCU 944 2469 UUUGAGACUCCUCAGUUCU 944 2491 AGAACUGAGGAGUCUCAAA 1167 2487 UUGCCACUUUUUUUUUUAA 945 2487 UUGCCACUUUUUUUUUUAA 945 2509 UUAAAAAAAAAAGUGGCAA 1168 2505 AUCUCCACCAGUCAUUUUU 946 2505 AUCUCCACCAGUCAUUUUU 946 2527 AAAAAUGACUGGUGGAGAU 1169 2523 UCAGACCUUUUAACUCCUC 947 2523 UCAGACCUUUUAACUCCUC 947 2545 GAGGAGUUAAAAGGUCUGA 1170 2541 CAAUUCCAACACUGAUUUC 948 2541 CAAUUCCAACACUGAUUUC 948 2563 GAAAUCAGUGUUGGAAUUG 1171 2559 CCCCUUUUGCAUUCUCCCU 949 2559 CCCCUUUUGCAUUCUCCCU 949 2581 AGGGAGAAUGCAAAAGGGG 1172 2577 UCCUUCCCUUCCUUGUAGC 950 2577 UCCUUCCCUUCCUUGUAGC 950 2599 GCUACAAGGAAGGGAAGGA 1173 2595 CCUUUUGACUUUCAUUGGA 951 2595 CCUUUUGACUUUCAUUGGA 951 2617 UCCAAUGAAAGUCAAAAGG 1174 2613 AAAUUAGGAUGUAAAUCUG 952 2613 AAAUUAGGAUGUAAAUCUG 952 2635 CAGAUUUACAUCCUAAUUU 1175 2631 GCUCAGGAGACCUGGAGGA 953 2631 GCUCAGGAGACCUGGAGGA 953 2653 UCCUCCAGGUCUCCUGAGC 1176 2649 AGCAGAGGAUAAUUAGCAU 954 2649 AGCAGAGGAUAAUUAGCAU 954 2671 AUGCUAAUUAUCCUCUGCU 1177 2667 UCUCAGGUUAAGUGUGAGU 955 2667 UCUCAGGUUAAGUGUGAGU 955 2689 ACUCACACUUAACCUGAGA 1178 2685 UAAUCUGAGAAACAAUGAC 956 2685 UAAUCUGAGAAACAAUGAC 956 2707 GUCAUUGUUUCUCAGAUUA 1179 2703 CUAAUUCUUGCAUAUUUUG 957 2703 CUAAUUCUUGCAUAUUUUG 957 2725 CAAAAUAUGCAAGAAUUAG 1180 2721 GUAACUUCCAUGUGAGGGU 958 2721 GUAACUUCCAUGUGAGGGU 958 2743 ACCCUCACAUGGAAGUUAC 1181 2739 UUUUCAGCAUUGAUAUUUG 959 2739 UUUUCAGCAUUGAUAUUUG 959 2761 CAAAUAUCAAUGCUGAAAA 1182 2757 GUGCAUUUUCUAAACAGAG 960 2757 GUGCAUUUUCUAAACAGAG 960 2779 CUCUGUUUAGAAAAUGCAC 1183 2775 GAUGAGGUGGUAUCUUCAC 961 2775 GAUGAGGUGGUAUCUUCAC 961 2797 GUGAAGAUACCACCUCAUC 1184 2793 CGUAGAACAUUGGUAUUCG 962 2793 CGUAGAACAUUGGUAUUCG 962 2815 CGAAUACCAAUGUUCUACG 1185 2811 GCUUGAGAAAAAAAGAAUA 963 2811 GCUUGAGAAAAAAAGAAUA 963 2833 UAUUCUUUUUUUCUCAAGC 1186 2829 AGUUGAACCUAUUUCUCUU 964 2829 AGUUGAACCUAUUUCUCUU 964 2851 AAGAGAAAUAGGUUCAACU 1187 2847 UUCUUUACAAGAUGGGUCC 965 2847 UUCUUUACAAGAUGGGUCC 965 2869 GGACCCAUCUUGUAAAGAA 1188 2865 CAGGAUUCCUCUUUUCUCU 966 2865 CAGGAUUCCUCUUUUCUCU 966 2887 AGAGAAAAGAGGAAUCCUG 1189 2883 UGCCAUAAAUGAUUAAUUA 967 2883 UGCCAUAAAUGAUUAAUUA 967 2905 UAAUUAAUCAUUUAUGGCA 1190 2901 AAAUAGCUUUUGUGUCUUA 968 2901 AAAUAGCUUUUGUGUCUUA 968 2923 UAAGACACAAAAGCUAUUU 1191 2919 ACAUUGGUAGCCAGCCAGC 969 2919 ACAUUGGUAGCCAGCCAGC 969 2941 GCUGGCUGGCUACCAAUGU 1192 2937 CCAAGGCUCUGUUUAUGCU 970 2937 CCAAGGCUCUGUUUAUGCU 970 2959 AGCAUAAACAGAGCCUUGG 1193 2955 UUUUGGGGGGCAUAUAUUG 971 2955 UUUUGGGGGGCAUAUAUUG 971 2977 CAAUAUAUGCCCCCCAAAA 1194 2973 GGGUUCCAUUCUCACCUAU 972 2973 GGGUUCCAUUCUCACCUAU 972 2995 AUAGGUGAGAAUGGAACCC 1195 2991 UCCACACAACAUAUCCGUA 973 2991 UCCACACAACAUAUCCGUA 973 3013 UACGGAUAUGUUGUGUGGA 1196 3009 AUAUAUCCCCUCUACUCUU 974 3009 AUAUAUCCCCUCUACUCUU 974 3031 AAGAGUAGAGGGGAUAUAU 1197 3027 UACUUCCCCCAAAUUUAAA 975 3027 UACUUCCCCCAAAUUUAAA 975 3049 UUUAAAUUUGGGGGAAGUA 1198 3045 AGAAGUAUGGGAAAUGAGA 976 3045 AGAAGUAUGGGAAAUGAGA 976 3067 UCUCAUUUCCCAUACUUCU 1199 3063 AGGCAUUUCCCCCACCCCA 977 3063 AGGCAUUUCCCCCACCCCA 977 3085 UGGGGUGGGGGAAAUGCCU 1200 3081 AUUUCUCUCCUCACACACA 978 3081 AUUUCUCUCCUCACACACA 978 3103 UGUGUGUGAGGAGAGAAAU 1201 3099 AGACUCAUAUUACUGGUAG 979 3099 AGACUCAUAUUACUGGUAG 979 3121 CUACCAGUAAUAUGAGUCU 1202 3117 GGAACUUGAGAACUUUAUU 980 3117 GGAACUUGAGAACUUUAUU 980 3139 AAUAAAGUUCUCAAGUUCC 1203 3135 UUCCAAGUUGUUCAAACAU 981 3135 UUCCAAGUUGUUCAAACAU 981 3157 AUGUUUGAACAACUUGGAA 1204 3153 UUUACCAAUCAUAUUAAUA 982 3153 UUUACCAAUCAUAUUAAUA 982 3175 UAUUAAUAUGAUUGGUAAA 1205 3171 ACAAUGAUGCUAUUUGCAA 983 3171 ACAAUGAUGCUAUUUGCAA 983 3193 UUGCAAAUAGCAUCAUUGU 1206 3189 AUUCCUGCUCCUAGGGGAG 984 3189 AUUCCUGCUCCUAGGGGAG 984 3211 CUCCCCUAGGAGCAGGAAU 1207 3207 GGGGAGAUAAGAAACCCUC 985 3207 GGGGAGAUAAGAAACCCUC 985 3229 GAGGGUUUCUUAUCUCCCC 1208 3225 CACUCUCUACAGGUUUGGG 986 3225 CACUCUCUACAGGUUUGGG 986 3247 CCCAAACCUGUAGAGAGUG 1209 3243 GUACAAGUGGCAACCUGCU 987 3243 GUACAAGUGGCAACCUGCU 987 3265 AGCAGGUUGCCACUUGUAC 1210 3261 UUCCAUGGCCGUGUAGAAG 988 3261 UUCCAUGGCCGUGUAGAAG 988 3283 CUUCUACACGGCCAUGGAA 1211 3279 GCAUGGUGCCCUGGCUUCU 989 3279 GCAUGGUGCCCUGGCUUCU 989 3301 AGAAGCCAGGGCACCAUGC 1212 3297 UCUGAGGAAGCUGGGGUUC 990 3297 UCUGAGGAAGCUGGGGUUC 990 3319 GAACCCCAGCUUCCUCAGA 1213 3315 CAUGACAAUGGCAGAUGUA 991 3315 CAUGACAAUGGCAGAUGUA 991 3337 UACAUCUGCCAUUGUCAUG 1214 3333 AAAGUUAUUCUUGAAGUCA 992 3333 AAAGUUAUUCUUGAAGUCA 992 3355 UGACUUCAAGAAUAACUUU 1215 3351 AGAUUGAGGCUGGGAGACA 993 3351 AGAUUGAGGCUGGGAGACA 993 3373 UGUCUCCCAGCCUCAAUCU 1216 3369 AGCCGUAGUAGAUGUUCUA 994 3369 AGCCGUAGUAGAUGUUCUA 994 3391 UAGAACAUCUACUACGGCU 1217 3387 ACUUUGUUCUGCUGUUCUC 995 3387 ACUUUGUUCUGCUGUUCUC 995 3409 GAGAACAGCAGAACAAAGU 1218 3405 CUAGAAAGAAUAUUUGGUU 996 3405 CUAGAAAGAAUAUUUGGUU 996 3427 AACCAAAUAUUCUUUCUAG 1219 3423 UUUCCUGUAUAGGAAUGAG 997 3423 UUUCCUGUAUAGGAAUGAG 997 3445 CUCAUUCCUAUACAGGAAA 1220 3441 GAUUAAUUCCUUUCCAGGU 998 3441 GAUUAAUUCCUUUCCAGGU 998 3463 ACCUGGAAAGGAAUUAAUC 1221 3459 UAUUUUAUAAUUCUGGGAA 999 3459 UAUUUUAUAAUUCUGGGAA 999 3481 UUCCCAGAAUUAUAAAAUA 1222 3477 AGCAAAACCCAUGCCUCCC 1000 3477 AGCAAAACCCAUGCCUCCC 1000 3499 GGGAGGCAUGGGUUUUGCU 1223 3495 CCCUAGCCAUUUUUACUGU 1001 3495 CCCUAGCCAUUUUUACUGU 1001 3517 ACAGUAAAAAUGGCUAGGG 1224 3513 UUAUCCUAUUUAGAUGGCC 1002 3513 UUAUCCUAUUUAGAUGGCC 1002 3535 GGCCAUCUAAAUAGGAUAA 1225 3531 CAUGAAGAGGAUGCUGUGA 1003 3531 CAUGAAGAGGAUGCUGUGA 1003 3553 UCACAGCAUCCUCUUCAUG 1226 3549 AAAUUCCCAACAAACAUUG 1004 3549 AAAUUCCCAACAAACAUUG 1004 3571 CAAUGUUUGUUGGGAAUUU 1227 3567 GAUGCUGACAGUCAUGCAG 1005 3567 GAUGCUGACAGUCAUGCAG 1005 3589 CUGCAUGACUGUCAGCAUC 1228 3585 GUCUGGGAGUGGGGAAGUG 1006 3585 GUCUGGGAGUGGGGAAGUG 1006 3607 CACUUCCCCACUCCCAGAC 1229 3603 GAUCUUUUGUUCCCAUCCU 1007 3603 GAUCUUUUGUUCCCAUCCU 1007 3625 AGGAUGGGAACAAAAGAUC 1230 3621 UCUUCUUUUAGCAGUAAAA 1008 3621 UCUUCUUUUAGCAGUAAAA 1008 3643 UUUUACUGCUAAAAGAAGA 1231 3639 AUAGCUGAGGGAAAAGGGA 1009 3639 AUAGCUGAGGGAAAAGGGA 1009 3661 UCCCUUUUCCCUCAGCUAU 1232 3657 AGGGAAAAGGAAGUUAUGG 1010 3657 AGGGAAAAGGAAGUUAUGG 1010 3679 CCAUAACUUCCUUUUCCCU 1233 3675 GGAAUACCUGUGGUGGUUG 1011 3675 GGAAUACCUGUGGUGGUUG 1011 3697 CAACCACCACAGGUAUUCC 1234 3693 GUGAUCCCUAGGUCUUGGG 1012 3693 GUGAUCCCUAGGUCUUGGG 1012 3715 CCCAAGACCUAGGGAUCAC 1235 3711 GAGCUCUUGGAGGUGUCUG 1013 3711 GAGCUCUUGGAGGUGUCUG 1013 3733 CAGACACCUCCAAGAGCUC 1236 3729 GUAUCAGUGGAUUUCCCAU 1014 3729 GUAUCAGUGGAUUUCCCAU 1014 3751 AUGGGAAAUCCACUGAUAC 1237 3747 UCCCCUGUGGGAAAUUAGU 1015 3747 UCCCCUGUGGGAAAUUAGU 1015 3769 ACUAAUUUCCCACAGGGGA 1238 3765 UAGGCUCAUUUACUGUUUU 1016 3765 UAGGCUCAUUUACUGUUUU 1016 3787 AAAACAGUAAAUGAGCCUA 1239 3783 UAGGUCUAGCCUAUGUGGA 1017 3783 UAGGUCUAGCCUAUGUGGA 1017 3805 UCCACAUAGGCUAGACCUA 1240 3801 AUUUUUUCCUAACAUACCU 1018 3801 AUUUUUUCCUAACAUACCU 1018 3823 AGGUAUGUUAGGAAAAAAU 1241 3819 UAAGCAAACCCAGUGUCAG 1019 3819 UAAGCAAACCCAGUGUCAG 1019 3841 CUGACACUGGGUUUGCUUA 1242 3837 GGAUGGUAAUUCUUAUUCU 1020 3837 GGAUGGUAAUUCUUAUUCU 1020 3859 AGAAUAAGAAUUACCAUCC 1243 3855 UUUCGUUCAGUUAAGUUUU 1021 3855 UUUCGUUCAGUUAAGUUUU 1021 3877 AAAACUUAACUGAACGAAA 1244 3873 UUCCCUUCAUCUGGGCACU 1022 3873 UUCCCUUCAUCUGGGCACU 1022 3895 AGUGCCCAGAUGAAGGGAA 1245 3891 UGAAGGGAUAUGUGAAACA 1023 3891 UGAAGGGAUAUGUGAAACA 1023 3913 UGUUUCACAUAUCCCUUCA 1246 3909 AAUGUUAACAUUUUUGGUA 1024 3909 AAUGUUAACAUUUUUGGUA 1024 3931 UACCAAAAAUGUUAACAUU 1247 3927 AGUCUUCAACCAGGGAUUG 1025 3927 AGUCUUCAACCAGGGAUUG 1025 3949 CAAUCCCUGGUUGAAGACU 1248 3945 GUUUCUGUUUAACUUCUUA 1026 3945 GUUUCUGUUUAACUUCUUA 1026 3967 UAAGAAGUUAAACAGAAAC 1249 3963 AUAGGAAAGCUUGAGUAAA 1027 3963 AUAGGAAAGCUUGAGUAAA 1027 3985 UUUACUCAAGCUUUCCUAU 1250 3981 AAUAAAUAUUGUCUUUUUG 1028 3981 AAUAAAUAUUGUCUUUUUG 1028 4003 CAAAAAGACAAUAUUUAUU 1251 3986 AUAUUGUCUUUUUGUAUGU 1029 3986 AUAUUGUCUUUUUGUAUGU 1029 4008 ACAUACAAAAAGACAAUAU 1252 The 3′-ends of the Upper sequence and the Lower sequence of the siNA construct can include an overhang sequence, for example about 1, 2, 3, or 4 nucleotides in length, preferably 2 nucleotides in length, wherein the overhanging sequence of the lower sequence is optionally complementary to a portion of the target sequence. The upper sequence is also referred to as the sense strand, whereas the lower sequence is also referred to as the antisense strand. The upper and lower sequences in the Table can further comprise a chemical modification having Formulae I-VII or any combination thereof.

TABLE III Interleukin and Interleukin receptor Synthetic Modified siNA constructs Target Seq Seq Pos Target ID Cmpd# Aliases Sequence ID IL2RG 118 ACACCACAGCUGAUUUCUUCCUG 1253 IL2RG:120U21 sense siNA ACCACAGCUGAUUUCUUCCTT 1311 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:132U21 sense siNA UUCUUCCUGACCACUAUGCTT 1312 138 CUGACCACUAUGCCCACUGACUC 1255 IL2RG:140U21 sense siNA GACCACUAUGCCCACUGACTT 1313 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:157U21 sense siNA ACUCCCUCAGUGUUUCCACTT 1314 262 CCAACCUCACUCUGCAUUAUUGG 1257 IL2RG:264U21 sense siNA AACCUCACUCUGCAUUAUUTT 1315 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:304U21 sense siNA AUAAAGUCCAGAAGUGCAGTT 1316 303 GAUAAAGUCCAGAAGUGCAGCCA 1259 IL2RG:305U21 sense siNA UAAAGUCCAGAAGUGCAGCTT 1317 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:346U21 sense siNA UCACUUCUGGCUGUCAGUUTT 1318 118 ACACCACAGCUGAUUUCUUCCUG 1253 IL2RG:138L21 antisense siNA GGAAGAAAUCAGCUGUGGUTT 1319 (120C) 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:150L21 antisense siNA GCAUAGUGGUCAGGAAGAATT 1320 (132C) 138 CUGACCACUAUGCCCACUGACUC 1255 IL2RG:158L21 antisense siNA GUCAGUGGGCAUAGUGGUCTT 1321 (140C) 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:175L21 antisense siNA GUGGAAACACUGAGGGAGUTT 1322 (157C) 262 CCAACCUCACUCUGCAUUAUUGG 1257 IL2RG:282L21 antisense siNA AAUAAUGCAGAGUGAGGUUTT 1323 (264C) 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:322L21 antisense siNA CUGCACUUCUGGACUUUAUTT 1324 (304C) 303 GAUAAAGUCCAGAAGUGCAGCCA 1259 IL2RG:323L21 antisense siNA GCUGCACUUCUGGACUUUATT 1325 (305C) 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:364L21 antisense siNA AACUGACAGCCAGAAGUGATT 1326 (346C) 118 ACACCACAGCUGAUUUCUUCCUG 1253 IL2RG:120U21 sense siNA B AccAcAGcuGAuuucuuccTT B 1327 stab04 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:132U21 sense siNA B uucuuccuGAccAcuAuGcTT B 1328 stab04 138 CUGACCACUAUGCCCACUGACUC 1255 IL2RG:140U21 sense siNA B GAccAcuAuGcccAcuGAcTT B 1329 stab04 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:157U21 sense siNA B AcucccucAGuGuuuccAcTT B 1330 stab04 262 CCAACCUCACUCUGCAUUAUUGG 1257 IL2RG:264U21 sense siNA B AAccucAcucuGcAuuAuuTT B 1331 stab04 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:304U21 sense siNA B AuAAAGuccAGAAGuGcAGTT B 1332 stab04 303 GAUAAAGUCCAGAAGUGCAGCCA 1259 IL2RG:305U21 sense siNA B uAAAGuccAGAAGuGcAGcTT B 1333 stab04 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:346U21 sense siNA B ucAcuucuGGcuGucAGuuTT B 1334 stab04 118 ACACCACAGCUGAUUUCUUCCUG 1253 IL2RG:138L21 antisense siNA GGAAGAAAucAGcuGuGGuTsT 1335 (120C) stab05 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:150L21 antisense siNA GcAuAGuGGucAGGAAGAATsT 1336 (132C) stab05 138 CUGACCACUAUGCCCACUGACUC 1255 IL2RG:158L21 antisense siNA GucAGuGGGcAuAGuGGucTsT 1337 (140C) stab05 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:175L21 antisense siNA GuGGAAAcAcuGAGGGAGuTsT 1338 (157C) stab05 262 CCAACCUCACUCUGCAUUAUUGG 1257 IL2RG:282L21 antisense siNA AAuAAuGcAGAGuGAGGuuTsT 1339 (264C) stab05 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:322L21 antisense siNA cuGcAcuucuGGAcuuuAuTsT 1340 (304C) stab05 303 GAUAAAGUCCAGAAGUGCAGCCA 1259 IL2RG:323L21 antisense siNA GcuGcAcuucuGGAcuuuATsT 1341 (305C) stab05 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:364L21 antisense siNA AAcuGAcAGccAGAAGuGATsT 1342 (346C) stab05 118 ACACCACAGCUGAUUUCUUCCUG 1253 IL2RG:120U21 sense siNA B AccAcAGcuGAuuucuuccTT B 1343 stab07 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:132U21 sense siNA B uucuuccuGAccAcuAuGcTT B 1344 stab07 138 CUGACCACUAUGCCCACUGACUC 1255 IL2RG:140U21 sense siNA B GAccAcuAuGcccAcuGAcTT B 1345 stab07 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:157U21 sense siNA B AcucccucAGuGuuuccAcTT B 1346 stab07 262 CCAACCUCACUCUGCAUUAUUGG 1257 IL2RG:264U21 sense siNA B AAccucAcucuGcAuuAuuTT B 1347 stab07 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:304U21 sense siNA B AuAAAGuccAGAAGuGcAGTT B 1348 stab07 303 GAUAAAGUCCAGAAGUGCAGCCA 1259 IL2RG:305U21 sense siNA B uAAAGuccAGAAGuGcAGcTT B 1349 stab07 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:346U21 sense siNA B ucAcuucuGGcuGucAGuuTT B 1350 stab07 118 ACACCACAGCUGAUUUCUUCCUG 1253 IL2RG:138L21 antisense siNA GGAAGAAAucAGcuGuGGuTsT 1351 (120C) stab11 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:150L21 antisense siNA GcAuAGuGGucAGGAAGAATsT 1352 (132C) stab11 138 CUGACCACUAUGCCCACUGACUC 1255 IL2RG:158L21 antisense siNA GucAGuGGGcAuAGuGGucTsT 1353 (140C) stab11 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:175L21 antisense siNA GuGGAAAcAcuGAGGGAGuTsT 1354 (157C) stab11 262 CCAACCUCACUCUGCAUUAUUGG 1257 IL2RG:282L21 antisense siNA AAuAAuGcAGAGuGAGGuuTsT 1355 (264C) stab11 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:322L21 antisense siNA cuGcAcuucuGGAcuuuAuTsT 1356 (304C) stab11 303 GAUAAAGUCCAGAAGUGCAGCCA 1259 IL2RG:323L21 antisense siNA GcuGcAcuucuGGAcuuuATsT 1357 (305C) stab11 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:364L21 antisense siNA AAcuGAcAGccAGAAGuGATsT 1358 (346C) stab11 118 ACACCACAGCUGAUUUCUUCCUG 1253 IL2RG:120U21 sense siNA B AccAcAGcuGAuuucuuccTT B 1359 stab18 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:132U21 sense siNA B uucuuccuGAccAcuAuGcTT B 1360 stab18 138 CUGACCACUAUGCCCACUGACUC 1255 IL2RG:140U21 sense siNA B GAccAcuAuGcccAcuGAcTT B 1361 stab18 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:157U21 sense siNA B AcucccucAGuGuuuccAcTT B 1362 stab18 262 CCAACCUCACUCUGCAUUAUUGG 1257 IL2RG:264U21 sense siNA B AAccucAcucuGcAuuAuuTT B 1363 stab18 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:304U21 sense siNA B AuAAAGuccAGAAGuGcAGTT B 1364 stab18 303 GAUAAAGUCCAGAAGUGCAGCCA 1259 IL2RG:305U21 sense siNA B uAAAGuccAGAAGuGcAGcTT B 1365 stab18 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:346U21 sense siNA B ucAcuucuGGcuGucAGuuTT B 1366 stab18 118 ACACCACAGCUGAUUUCUUCCUG 1253 IL2RG:138L21 antisense siNA GGAAGAAAucAGcuGuGGuTsT 1367 (120C) stab08 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:150L21 antisense siNA GcAuAGuGGucAGGAAGAATsT 1368 (132C) stab08 138 CUGACCACUAUGCCCACUGACUC 1255 IL2RG:158L21 antisense siNA GucAGuGGGcAuAGuGGucTsT 1369 (140C) stab08 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:175L21 antisense siNA GuGGAAAcAcuGAGGGAGuTsT 1370 (157C) stab08 262 CCAACCUCACUCUGCAUUAUUGG 1257 IL2RG:282L21 antisense siNA AAuAAuGcAGAGuGAGGuuTsT 1371 (264C) stab08 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:322L21 antisense siNA cuGcAcuucuGGAcuuuAuTsT 1372 (304C) stab08 303 GAUAAAGUCCAGAAGUGCAGCCA 1259 IL2RG:323L21 antisense siNA GcuGcAcuucuGGAcuuuATsT 1373 (305C) stab08 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:364L21 antisense siNA AAcuGAcAGccAGAAGuGATsT 1374 (346C) stab08 118 ACACCACAGCUGAUUUCUUCCUG 1253 IL2RG:120U21 sense siNA B ACCACAGCUGAUUUCUUCCTT B 1375 stab09 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:132U21 sense siNA B UUCUUCCUGACCACUAUGCTT B 1376 stab09 138 CUGACCACUAUGCCCACUGACUC 1255 IL2RG:140U21 sense siNA B GACCACUAUGCCCACUGACTT B 1377 stab09 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:157U21 sense siNA B ACUCCCUCAGUGUUUCCACTT B 1378 stab09 262 CCAACCUCACUCUGCAUUAUUGG 1257 IL2RG:264U21 sense siNA B AACCUCACUCUGCAUUAUUTT B 1379 stab09 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:304U21 sense siNA B AUAAAGUCCAGAAGUGCAGTT B 1380 stab09 303 GAUAAAGUCCAGAAGUGCAGCCA 1259 IL2RG:305U21 sense siNA B UAAAGUCCAGAAGUGCAGCTT B 1381 stab09 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:346U21 sense siNA B UCACUUCUGGCUGUCAGUUTT B 1382 stab09 118 ACACCACAGCUGAUUUCUUCCUG 1253 IL2RG:138L21 antisense siNA GGAAGAAAUCAGCUGUGGUTsT 1383 (120C) stab10 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:150L21 antisense siNA GCAUAGUGGUCAGGAAGAATsT 1384 (132C) stab10 138 CUGACCACUAUGCCCACUGACUC 1255 IL2RG:158L21 antisense siNA GUCAGUGGGCAUAGUGGUCTsT 1385 (140C) stab10 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:175L21 antisense siNA GUGGAAACACUGAGGGAGUTsT 1386 (157C) stab10 262 CCAACCUCACUCUGCAUUAUUGG 1257 IL2RG:282L21 antisense siNA AAUAAUGCAGAGUGAGGUUTsT 1387 (264C) stab10 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:322L21 antisense siNA CUGCACUUCUGGACUUUAUTsT 1388 (304C) stab10 303 GAUAAAGUCCAGAAGUGCAGCCA 1259 IL2RG:323L21 antisense siNA GCUGCACUUCUGGACUUUATsT 1389 (305C) stab10 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:364L21 antisense siNA AACUGACAGCCAGAAGUGATsT 1390 (346C) stab10 118 ACACCACAGCUGAUUUCUUCCUG 1253 IL2RG:138L21 antisense siNA GGAAGAAAucAGcuGuGGuTT B 1391 (120C) stab19 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:150L21 antisense siNA GcAuAGuGGucAGGAAGAATT B 1392 (132C) stab19 138 CUGACCACUAUGCCCACUGACUC 1255 IL2RG:158L21 antisense siNA GucAGuGGGcAuAGuGGucTT B 1393 (140C) stab19 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:175L21 antisense siNA GuGGAAAcAcuGAGGGAGuTT B 1394 (157C) stab19 262 CCAACCUCACUCUGCAUUAUUGG 1257 IL2RG:282L21 antisense siNA AAuAAuGcAGAGuGAGGuuTT B 1395 (264C) stab19 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:322L21 antisense siNA cuGcAcuucuGGAcuuuAuTT B 1396 (304C) stab19 303 GAUAAAGUCCAGAAGUGCAGCCA 1259 IL2RG:323L21 antisense siNA GcuGcAcuucuGGAcuuuATT B 1397 (305C) stab19 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:364L21 antisense siNA AAcuGAcAGccAGAAGuGATT B 1398 (346C) stab19 118 ACACCACAGCUGAUUUCUUCCUG 1253 IL2RG:138L21 antisense siNA GGAAGAAAUCAGCUGUGGUTT B 1399 (120C) stab22 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:150L21 antisense siNA GCAUAGUGGUCAGGAAGAATT B 1400 (132C) stab22 138 CUGACCACUAUGCCCACUGACUC 1255 IL2RG:158L21 antisense siNA GUCAGUGGGCAUAGUGGUCTT B 1401 (140C) stab22 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:175L21 antisense siNA GUGGAAACACUGAGGGAGUTT B 1402 (157C) stab22 262 CCAACCUCACUCUGCAUUAUUGG 1257 IL2RG:282L21 antisense siNA AAUAAUGCAGAGUGAGGUUTT B 1403 (264C) stab22 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:322L21 antisense siNA CUGCACUUCUGGACUUUAUTT B 1404 (304C) stab22 303 GAUAAAGUCCAGAAGUGCAGCCA 1259 IL2RG:323L21 antisense siNA GCUGCACUUCUGGACUUUATT B 1405 (305C) stab22 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:364L21 antisense siNA AACUGACAGCCAGAAGUGATT B 1406 (346C) stab22 IL4 487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:489U21 sense siNA GCCUCACAGAGCAGAAGACTT 1407 489 GCCUCACAGAGCAGAAGACUCUG 1270 IL4:491U21 sense siNA CUCACAGAGCAGAAGACUCTT 1408 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:518U21 sense siNA GAGUUGACCGUAACAGACATT 1409 526 CGUAACAGACAUCUUUGCUGCCU 1272 IL4:528U21 sense siNA UAACAGACAUCUUUGCUGCTT 1410 545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:547U21 sense siNA CUCCAAGAACACAACUGAGTT 1411 606 UCUACAGCCACCAUGAGAAGGAC 1274 IL4:608U21 sense siNA UACAGCCACCAUGAGAAGGTT 1412 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:730U21 sense siNA GAAUUCCUGUCCUGUGAAGTT 1413 745 GAAGGAAGCCAACCAGAGUACGU 1276 IL4:747U21 sense siNA AGGAAGCCAACCAGAGUACTT 1414 487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:507L21 antisense siNA GUCUUCUGCUCUGUGAGGCTT 1415 (489C) 489 GCCUCACAGAGCAGAAGACUCUG 1270 IL4:509L21 antisense siNA GAGUCUUCUGCUCUGUGAGTT 1416 (491C) 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:536L21 antisense siNA UGUCUGUUACGGUCAACUCTT 1417 (518C) 526 CGUAACAGACAUCUUUGCUGCCU 1272 IL4:546L21 antisense siNA GCAGCAAAGAUGUCUGUUATT 1418 (528C) 545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:565L21 antisense siNA CUCAGUUGUGUUCUUGGAGTT 1419 (547C) 606 UCUACAGCCACCAUGAGAAGGAC 1274 IL4:626L21 antisense siNA CCUUCUCAUGGUGGCUGUATT 1420 (608C) 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:748L21 antisense siNA CUUCACAGGACAGGAAUUCTT 1421 (730C) 745 GAAGGAAGCCAACCAGAGUACGU 1276 IL4:765L21 antisense siNA GUACUCUGGUUGGCUUCCUTT 1422 (747C) 487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:489U21 sense siNA B GccucAcAGAGcAGAAGAcTT B 1423 stab04 489 GCCUCACAGAGCAGAAGACUCUG 1270 IL4:491U21 sense siNA B cucAcAGAGcAGAAGAcucTT B 1424 stab04 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:518U21 sense siNA B GAGuuGAccGuAAcAGAcATT B 1425 stab04 526 CGUAACAGACAUCUUUGCUGCCU 1272 IL4:528U21 sense siNA B uAAcAGAcAucuuuGcuGcTT B 1426 stab04 545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:547U21 sense siNA B cuccAAGAAcAcAAcuGAGTT B 1427 stab04 606 UCUACAGCCACCAUGAGAAGGAC 1274 IL4:608U21 sense siNA B uAcAGccAccAuGAGAAGGTT B 1428 stab04 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:730U21 sense siNA B GAAuuccuGuccuGuGAAGTT B 1429 stab04 745 GAAGGAAGCCAACCAGAGUACGU 1276 IL4:747U21 sense siNA B AGGAAGccAAccAGAGuAcTT B 1430 stab04 487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:507L21 antisense siNA GucuucuGcucuGuGAGGcTsT 1431 (489C) stab05 489 GCCUCACAGAGCAGAAGACUCUG 1270 IL4:509L21 antisense siNA GAGucuucuGcucuGuGAGTsT 1432 (491C) stab05 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:536L21 antisense siNA uGucuGuuAcGGucAAcucTsT 1433 (518C) stab05 526 CGUAACAGACAUCUUUGCUGCCU 1272 IL4:546L21 antisense siNA GcAGcAAAGAuGucuGuuATsT 1434 (528C) stab05 545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:565L21 antisense siNA cucAGuuGuGuucuuGGAGTsT 1435 (547C) stab05 606 UCUACAGCCACCAUGAGAAGGAC 1274 IL4:626L21 antisense siNA ccuucucAuGGuGGcuGuATsT 1436 (608C) stab05 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:748L21 antisense siNA cuucAcAGGAcAGGAAuucTsT 1437 (730C) stab05 745 GAAGGAAGCCAACCAGAGUACGU 1276 IL4:765L21 antisense siNA GuAcucuGGuuGGcuuccuTsT 1438 (747C) stab05 487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:489U21 sense siNA B GccucAcAGAGcAGAAGAcTT B 1439 stab07 489 GCCUCACAGAGCAGAAGACUCUG 1270 IL4:491U21 sense siNA B cucAcAGAGcAGAAGAcucTT B 1440 stab07 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:518U21 sense siNA B GAGuuGAccGuAAcAGAcATT B 1441 stab07 526 CGUAACAGACAUCUUUGCUGCCU 1272 IL4:528U21 sense siNA B uAAcAGAcAucuuuGcuGcTT B 1442 stab07 545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:547U21 sense siNA B cuccAAGAAcAcAAcuGAGTT B 1443 stab07 606 UCUACAGCCACCAUGAGAAGGAC 1274 IL4:608U21 sense siNA B uAcAGccAccAuGAGAAGGTT B 1444 stab07 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:730U21 sense siNA B GAAuuccuGuccuGuGAAGTT B 1445 stab07 745 GAAGGAAGCCAACCAGAGUACGU 1276 IL4:747U21 sense siNA B AGGAAGccAAccAGAGuAcTT B 1446 stab07 487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:507L21 antisense siNA GucuucuGcucuGuGAGGcTsT 1447 (489C) stab11 489 GCCUCACAGAGCAGAAGACUCUG 1270 IL4:509L21 antisense siNA GAGucuucuGcucuGuGAGTsT 1448 (491C) stab11 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:536L21 antisense siNA uGucuGuuAcGGucAAcucTsT 1449 (518C) stab11 526 CGUAACAGACAUCUUUGCUGCCU 1272 IL4:546L21 antisense siNA GcAGcAAAGAuGucuGuuATsT 1450 (528C) stab11 545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:565L21 antisense siNA cucAGuuGuGuucuuGGAGTsT 1451 (547C) stab11 606 UCUACAGCCACCAUGAGAAGGAC 1274 IL4:626L21 antisense siNA ccuucucAuGGuGGcuGuATsT 1452 (608C) stab11 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:748L21 antisense siNA cuucAcAGGAcAGGAAuucTsT 1453 (730C) stab11 745 GAAGGAAGCCAACCAGAGUACGU 1276 IL4:765L21 antisense siNA GuAcucuGGuuGGcuuccuTsT 1454 (747C)stab11 487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:489U21 sense siNA B GccucAcAGAGcAGAAGAcTT B 1455 stab18 489 GCCUCACAGAGCAGAAGACUCUG 1270 IL4:491U21 sense siNA B cucAcAGAGcAGAAGAcucTT B 1456 stab18 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:518U21 sense siNA B GAGuuGAccGuAAcAGAcATT B 1457 stab18 526 CGUAACAGACAUCUUUGCUGCCU 1272 IL4:528U21 sense siNA B uAAcAGAcAucuuuGcuGcTT B 1458 stab18 545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:547U21 sense siNA B cuccAAGAAcAcAAcuGAGTT B 1459 stab18 606 UCUACAGCCACCAUGAGAAGGAC 1274 IL4:608U21 sense siNA B uAcAGccAccAuGAGAAGGTT B 1460 stab18 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:730U21 sense siNA B GAAuuccuGuccuGuGAAGTT B 1461 stab18 745 GAAGGAAGCCAACCAGAGUACGU 1276 IL4:747U21 sense siNA B AGGAAGccAAccAGAGuAcTT B 1462 stab18 487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:507L21 antisense siNA GucuucuGcucuGuGAGGcTsT 1463 (489C) stab08 489 GCCUCACAGAGCAGAAGACUCUG 1270 IL4:509L21 antisense siNA GAGucuucuGcucuGuGAGTsT 1464 (491C) stab08 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:536L21 antisense siNA uGucuGuuAcGGucAAcucTsT 1465 (518C) stab08 526 CGUAACAGACAUCUUUGCUGCCU 1272 IL4:546L21 antisense siNA GcAGcAAAGAuGucuGuuATsT 1466 (528C) stab08 545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:565L21 antisense siNA cucAGuuGuGuucuuGGAGTsT 1467 (547C) stab08 606 UCUACAGCCACCAUGAGAAGGAC 1274 IL4:626L21 antisense siNA ccuucucAuGGuGGcuGuATsT 1468 (608C) stab08 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:748L21 antisense siNA cuucAcAGGAcAGGAAuucTsT 1469 (730C) stabo8 745 GAAGGAAGCCAACCAGAGUACGU 1276 IL4:765L21 antisense siNA GuAcucuGGuuGGcuuccuTsT 1470 (747C) stab08 487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:489U21 sense siNA B GCCUCACAGAGCAGAAGACTT B 1471 stab09 489 GCCUCACAGAGCAGAAGACUCUG 1270 IL4:491U21 sense siNA B CUCACAGAGCAGAAGACUCTT B 1472 stab09 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:518U21 sense siNA B GAGUUGACCGUAACAGACATT B 1473 stab09 526 CGUAACAGACAUCUUUGCUGCCU 1272 IL4:528U21 sense siNA B UAACAGACAUCUUUGCUGCTT B 1474 stab09 545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:547U21 sense siNA B CUCCAAGAACACAACUGAGTT B 1475 stab09 606 UCUACAGCCACCAUGAGAAGGAC 1274 IL4:608U21 sense siNA B UACAGCCACCAUGAGAAGGTT B 1476 stab09 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:730U21 sense siNA B GAAUUCCUGUCCUGUGAAGTT B 1477 stab09 745 GAAGGAAGCCAACCAGAGUACGU 1276 IL4:747U21 sense siNA B AGGAAGCCAACCAGAGUACTT B 1478 stab09 487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:507L21 antisense siNA GUCUUCUGCUCUGUGAGGCTsT 1479 (489C) stab10 489 GCCUCACAGAGCAGAAGACUCUG 1270 IL4:509L21 antisense siNA GAGUCUUCUGCUCUGUGAGTsT 1480 (491C) stab10 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:536L21 antisense siNA UGUCUGUUACGGUCAACUCTsT 1481 (518C) stab10 526 CGUAACAGACAUCUUUGCUGCCU 1272 IL4:546L21 antisense siNA GCAGCAAAGAUGUCUGUUATsT 1482 (528C) stab10 545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:565L21 antisense siNA CUCAGUUGUGUUCUUGGAGTsT 1483 (547C) stab10 606 UCUACAGCCACCAUGAGAAGGAC 1274 IL4:626L21 antisense siNA CCUUCUCAUGGUGGCUGUATsT 1484 (608C) stab10 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:748L21 antisense siNA CUUCACAGGACAGGAAUUCTsT 1485 (730C) stab10 745 GAAGGAAGCCAACCAGAGUACGU 1276 IL4:765L21 antisense siNA GUACUCUGGUUGGCUUCCUTsT 1486 (747C) stab10 487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:507L21 antisense siNA GucuucuGcucuGuGAGGcTT B 1487 (489C) stab19 489 GCCUCACAGAGCAGAAGACUCUG 1270 IL4:509L21 antisense siNA GAGucuucuGcucuGuGAGTT B 1488 (491C) stab19 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:536L21 antisense siNA uGucuGuuAcGGucAAcucTT B 1489 (518C) stab19 526 CGUAACAGACAUCUUUGCUGCCU 1272 IL4:546L21 antisense siNA GcAGcAAAGAuGucuGuuATT B 1490 (528C) stab19 545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:565L21 antisense siNA cucAGuuGuGuucuuGGAGTT B 1491 (547C) stab19 606 UCUACAGCCACCAUGAGAAGGAC 1274 IL4:626L21 antisense siNA ccuucucAuGGuGGcuGuATT B 1492 (6C8C) stab19 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:748L21 antisense siNA cuucAcAGGAcAGGAAuucTT B 1493 (730C) stab19 745 GAAGGAAGCCAACCAGAGUACGU 1276 IL4:765L21 antisense siNA GuAcucuGGuuGGcuuccuTT B 1494 (747C) stab19 487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:507L21 antisense siNA GUCUUCUGCUCUGUGAGGCTT B 1495 (489C) stab22 489 GCCUCACAGAGCAGAAGACUCUG 1270 IL4:509L21 antisense siNA GAGUCUUCUGCUCUGUGAGTT B 1496 (491C) stab22 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:536L21 antisense siNA UGUCUGUUACGGUCAACUCTT B 1497 (518C) stab22 526 CGUAACAGACAUCUUUGCUGCCU 1272 IL4:546L21 antisense siNA GCAGCAAAGAUGUCUGUUATT B 1498 (528C) stab22 545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:565L21 antisense siNA CUCAGUUGUGUUCUUGGAGTT B 1499 (547C) stab22 606 UCUACAGCCACCAUGAGAAGGAC 1274 IL4:626L21 antisense siNA CCUUCUCAUGGUGGCUGUATT B 1500 (608C) stab22 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:748L21 antisense siNA CUUCACAGGACAGGAAUUCTT B 1501 (730C) stab22 745 GAAGGAAGCCAACCAGAGUACGU 1276 IL4:765L21 antisense siNA GUACUCUGGUUGGCUUCCUTT B 1502 (747C) stab22 IL4R 469 CUAUACACUGGACCUGUGGGCUG 1277 IL4R:471U21 sense siNA AUACACUGGACCUGUGGGCTT 1503 551 CCAGGAAACCUGACAGUUCACAC 1278 IL4R:553U21 sense siNA AGGAAACCUGACAGUUCACTT 1504 1119 AGCACAACAUGAAAAGGGAUGAA 1279 IL4R:1121U21 sense siNA CACAACAUGAAAAGGGAUGTT 1505 1120 GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1122U21 sense siNA ACAACAUGAAAAGGGAUGATT 1506 1132 AAGGGAUGAAGAUCCUCACAAGG 1281 IL4R:1134U21 sense siNA GGGAUGAAGAUCCUCACAATT 1507 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3132U21 sense siNA GGGAAAUCGAUGAGAAAUUTT 1508 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283 IL4R:3133U21 sense siNA GGAAAUCGAUGAGAAAUUGTT 1509 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3171U21 sense siNA AUUGCCUAGAGGUGCUCAUTT 1510 469 CUAUACACUGGACCUGUGGGCUG 1277 IL4R:489L21 antisense siNA GCCCACAGGUCCAGUGUAUTT 1511 (471C) 551 CCAGGAAACCUGACAGUUCACAC 1278 IL4R:57IL21 antisense siNA GUGAACUGUCAGGUUUCCUTT 1512 (553C) 1119 AGCACAACAUGAAAAGGGAUGAA 1279 IL4R:1139L21 antisense siNA CAUCCCUUUUCAUGUUGUGTT 1513 (1121C) 1120 GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1140L21 antisense siNA UCAUCCCUUUUCAUGUUGUTT 1514 (1122C) 1132 AAGGGAUGAAGAUCCUCACAAGG 1281 IL4R:1152L21 antisense siNA UUGUGAGGAUCUUCAUCCCTT 1515 (1134C) 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3150L21 antisense siNA AAUUUCUCAUCGAUUUCCCTT 1516 (3132C) 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283 IL4R:315IL21 antisense siNA CAAUUUCUCAUCGAUUUCCTT 1517 (3133C) 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3189L21 antisense siNA AUGAGCACCUCUAGGCAAUTT 1518 (3171C) 469 CUAUACACUGGACCUGUGGGCUG 1277 IL4R:471U21 sense siNA B AuAcAcuGGAccuGuGGGcTT B 1519 stab04 551 CCAGGAAACCUGACAGUUCACAC 1278 IL4R:553U21 sense siNA B AGGAAAccuGAcAGuucAcTT B 1520 stab04 1119 AGCACAACAUGAAAAGGGAUGAA 1279 IL4R:1121U21 sense siNA B cAcAAcAuGAAAAGGGAuGTT B 1521 stab04 1120 GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1122U21 sense siNA B AcAAcAuGAAAAGGGAuGATT B 1522 stab04 1132 AAGGGAUGAAGAUCCUCACAAGG 1281 IL4R:1134U21 sense siNA B GGGAuGAAGAuccucAcAATT B 1523 stab04 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3132U21 sense siNA B GGGAAAucGAuGAGAAAuuTT B 1524 stab04 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283 IL4R:3133U21 sense siNA B GGAAAucGAuGAGAAAuuGTT B 1525 stab04 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3171U21 sense siNA B AuuGccuAGAGGuGcucAuTT B 1526 stab04 469 CUAUACACUGGACCUGUGGGCUG 1277 IL4R:489L21 antisense siNA GcccAcAGGuccAGuGuAuTsT 1527 (471C) stab05 551 CCAGGAAACCUGACAGUUCACAC 1278 IL4R:571L21 antisense siNA GuGAAcuGucAGGuuuccuTsT 1528 (553C) stab05 1119 AGCACAACAUGAAAAGGGAUGAA 1279 IL4R:1139L21 antisense siNA cAucccuuuucAuGuuGuGTsT 1529 (1121C) stab05 1120 GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1140L21 antisense siNA ucAucccuuuucAuGuuGuTsT 1530 (1122C) stab05 1132 AAGGGAUGAAGAUCCUCACAAGG 1281 IL4R:1152L21 antisense siNA uuGuGAGGAucuucAucccTsT 1531 (1134C) stab05 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3150L21 antisense siNA AAuuucucAucGAuuucccTsT 1532 (3132C) stab05 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283 IL4R:315IL21 antisense siNA cAAuuucucAucGAuuuccTsT 1533 (3133C) stab05 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3189L21 antisense siNA AuGAGcAccucuAGGcAAuTsT 1534 (3171C) stab05 469 CUAUACACUGGACCUGUGGGCUG 1277 IL4R:471U21 sense siNA B AuAcAcuGGAccuGuGGGcTT B 1535 stab07 551 CCAGGAAACCUGACAGUUCACAC 1278 IL4R:553U21 sense siNA B AGGAAAccuGAcAGuucAcTT B 1536 stab07 1119 AGCACAACAUGAAAAGGGAUGAA 1279 IL4R:1121U21 sense siNA B cAcAAcAuGAAAAGGGAuGTT B 1537 stab07 1120 GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1122U21 sense siNA B AcAAcAuGAAAAGGGAuGATT B 1538 stab07 1132 AAGGGAUGAAGAUCCUCACAAGG 1281 IL4R:1134U21 sense siNA B GGGAuGAAGAuccucAcAATT B 1539 stab07 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3132U21 sense siNA B GGGAAAucGAuGAGAAAuuTT B 1540 stab07 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283 IL4R:3133U21 sense siNA B GGAAAucGAuGAGAAAuuGTT B 1541 stab07 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3171U21 sense siNA B AuuGccuAGAGGuGcucAuTT B 1542 stab07 469 CUAUACACUGGACCUGUGGGCUG 1277 IL4R:489L21 antisense siNA GcccAcAGGuccAGuGuAuTsT 1543 (471C) stab11 551 CCAGGAAACCUGACAGUUCACAC 1278 IL4R:571L21 antisense siNA GuGAAcuGucAGGuuuccuTsT 1544 (553C) stab11 1119 AGCACAACAUGAAAAGGGAUGAA 1279 IL4R:1139L21 antisense siNA cAucccuuuucAuGuuGuGTsT 1545 (1121C) stab11 1120 GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1140L21 antisense siNA ucAucccuuuucAuGuuGuTsT 1546 (1122C) stab11 1132 AAGGGAUGAAGAUCCUCACAAGG 1281 IL4R:1152L21 antisense siNA uuGuGAGGAucuucAucccTsT 1547 (1134C) stab11 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3150L21 antisense siNA AAuuucucAl uc GAuuucccTsT 1548 (3132C) stab11 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283 IL4R:315IL21 antisense siNA cAAuuucucAucGAuuuccTsT 1549 (3133C) stab11 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3189L21 antisense siNA AuGAGcAccucuAGGcAAuTsT 1550 (3171C) stab11 469 CUAUACACUGGACCUGUGGGCUG 1277 IL4R:471U21 sense siNA B AuAcAcuGGAccuGuGGGcTTB 1551 stab18 551 CCAGGAAACCUGACAGUUCACAC 1278 IL4R:553U21 sense siNA B AGGAAAccuGAcAGuucAcTT B 1552 stab18 1119 AGCACAACAUGAAAAGGGAUGAA 1279 IL4R:1121U21 sense siNA B cAcAAcAuGAAAAGGGAuGTT B 1553 stab18 1120 GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1122U21 sense siNA B AcAAcAuGAAAAGGGAuGATT B 1554 stab18 1132 AAGGGAUGAAGAUCCUCACAAGG 1281 IL4R:1134U21 sense siNA B GGGAuGAAGAuccucAcAATT B 1555 stab18 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3132U21 sense siNA B GGGAAAucGAuGAGAAAuuTT B 1556 stab18 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283 IL4R:3133U21 sense siNA B GGAAAucGAuGAGAAAuuGTT B 1557 stab18 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3171U21 sense siNA B AuuGccuAGAGGuGcucAuTT B 1558 stab18 469 CUAUACACUGGACCUGUGGGCUG 1277 IL4R:489L21 antisense siNA GcccAcAGGuccAGuGuAuTsT 1559 (471C) stab08 551 CCAGGAAACCUGACAGUUCACAC 1278 IL4R:571L21 antisense siNA GuGAAcuGucAGGuuuccuTsT 1560 (553C) stab08 1119 AGCACAACAUGAAAAGGGAUGAA 1279 IL4R:1139L21 antisense siNA cAucccuuuucAuGuuGuGTsT 1561 (1121C) stab08 1120 GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1140L21 antisense siNA ucAucccuuuucAuGuuGuTsT 1562 (1122C) stab08 1132 AAGGGAUGAAGAUCCUCACAAGG 1281 IL4R:1152L21 antisense siNA uuGuGAGGAucuucAucccTsT 1563 (1134C) stab08 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3150L21 antisense siNA AAuuucucAucGAuuucccTsT 1564 (3132C) stab08 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283 IL4R:315IL21 antisense siNA cAAuuucucAucGAuuuccTsT 1565 (3133C) stab08 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3189L21 antisense siNA AuGAGcAccucuAGGcAAuTsT 1566 (3171C) stab08 469 CUAUACACUGGACCUGUGGGCUG 1277 36729 IL4R:471U21 sense siNA B AUACACUGGACCUGUGGGCTT B 1567 stab09 551 CCAGGAAACCUGACAGUUCACAC 1278 36730 IL4R:553U21 sense siNA B AGGAAACCUGACAGUUCACTT B 1568 stab09 1119 AGCACAACAUGAAAAGGGAUGAA 1279 36731 IL4R:1121U21 sense siNA B CACAACAUGAAAAGGGAUGTT B 1569 stab09 1120 GCACAACAUGAAAAGGGAUGAAG 1280 36732 IL4R:1122U21 sense siNA B ACAACAUGAAAAGGGAUGATT B 1570 stab09 1132 AAGGGAUGAAGAUCCUCACAAGG 1281 36733 IL4R:1134U21 sense siNA B GGGAUGAAGAUCCUCACAATT B 1571 stab09 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 36734 IL4R:3132U21 sense siNA B GGGAAAUCGAUGAGAAAUUTT B 1572 stab09 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283 36735 IL4R:3133U21 sense siNA B GGAAAUCGAUGAGAAAUUGTT B 1573 stab09 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 36736 IL4R:3171U21 sense siNA B AUUGCCUAGAGGUGCUCAUTT B 1574 stab09 469 CUAUACACUGGACCUGUGGGCUG 1277 IL4R:489L21 antisense siNA GCCCACAGGUCCAGUGUAUTsT 1575 (471C) stab10 551 CCAGGAAACCUGACAGUUCACAC 1278 IL4R:571L21 antisense siNA GUGAACUGUCAGGUUUCCUTsT 1576 (553C) stab10 1119 AGCACAACAUGAAAAGGGAUGAA 1279 IL4R:1139L21 antisense siNA CAUCCCUUUUCAUGUUGUGTsT 1577 (1121C) stab10 1120 GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1140L21 antisense siNA UCAUCCCUUUUCAUGUUGUTsT 1578 (1122C) stab10 1132 AAGGGAUGAAGAUCCUCACAAGG 1281 IL4R:1152L21 antisense siNA UUGUGAGGAUCUUCAUCCCTsT 1579 (1134C) stab10 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3150L21 antisense siNA AAUUUCUCAUCGAUUUCCCTsT 1580 (3132C) stab10 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283 IL4R:315IL21 antisense siNA CAAUUUCUCAUCGAUUUCCTsT 1581 (3133C) stab10 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3189L21 antisense siNA AUGAGCACCUCUAGGCAAUTsT 1582 (3171C) stab10 469 CUAUACACUGGACCUGUGGGCUG 1277 36737 IL4R:489L21 antisense siNA GcccAcAGGuccAGuGuAuTT B 1583 (471C) stab19 551 CCAGGAAACCUGACAGUUCACAC 1278 36738 IL4R:571L21 antisense siNA GuGAAcuGucAGGuuuccuTT B 1584 (553C) stab19 1119 AGCACAACAUGAAAAGGGAUGAA 1279 36739 IL4R:1139L21 antisense siNA cAucccuuuucAuGuuGuGTT B 1585 (1121C) stab19 1120 GCACAACAUGAAAAGGGAUGAAG 1280 36740 IL4R:1140L21 antisense siNA ucAucccuuuucAuGuuGuTT B 1586 (1122C) stab19 1132 AAGGGAUGAAGAUCCUCACAAGG 1281 36741 IL4R:1152L21 antisense siNA uuGuGAGGAucuucAucccTT B 1587 (1134C) stab19 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 36742 IL4R:3150L21 antisense siNA AAuuucucAucGAuuucccTT B 1588 (3132C) stab19 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283 36743 IL4R:315IL21 antisense siNA cAAuuucucAucGAuuuccTT B 1589 (3133C) stab19 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 36744 IL4R:3189L21 antisense siNA AuGAGcAccucuAGGcAAuTT B 1590 (3171C) stab19 469 CUAUACACUGGACCUGUGGGCUG 1277 36745 IL4R:489L21 antisense siNA GCCCACAGGUCCAGUGUAUTT B 1591 (471C) stab22 551 CCAGGAAACCUGACAGUUCACAC 1278 36746 IL4R:571L21 antisense siNA GUGAACUGUCAGGUUUCCUTT B 1592 (553C) stab22 1119 AGCACAACAUGAAAAGGGAUGAA 1279 36747 IL4R:1139L21 antisense siNA CAUCCCUUUUCAUGUUGUGTTB 1593 (1121C) stab22 1120 GCACAACAUGAAAAGGGAUGAAG 1280 36748 IL4R:1140L21 antisense siNA UCAUCCCUUUUCAUGUUGUTT B 1594 (1122C) stab22 1132 AAGGGAUGAAGAUCCUCACAAGG 1281 36749 IL4R:1152L21 antisense siNA UUGUGAGGAUCUUCAUCCCTT B 1595 (1134C) stab22 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 36750 IL4R:3150L21 antisense siNA AAUUUCUCAUCGAUUUCCCTT B 1596 (3132C) stab22 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283 36751 IL4R:315IL21 antisense siNA CAAUUUCUCAUCGAUUUCCTT B 1597 (3133C) stab22 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 36752 IL4R:3189L21 antisense siNA AUGAGCACCUCUAGGCAAUTT B 1598 (3171C) stab22 IL13 391 CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:393U21 sense siNA CAGUUUGUAAAGGACCUGCTT 1599 797 CACUUCACACACAGGCAACUGAG 1286 IL13:799U21 sense siNA CUUCACACACAGGCAACUGTT 1600 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:834U21 sense siNA AGGCACACUUCUUCUUGGUTT 1601 911 AAGACUGUGGCUGCUAGCACUUG 1288 IL13:913U21 sense siNA GACUGUGGCUGCUAGCACUTT 1602 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:965U21 sense siNA CACUAAAGCAGUGGACACCTT 1603 965 CACUAAAGCAGUGGACACCAGGA 1290 IL13:967U21 sense siNA CUAAAGCAGUGGACACCAGTT 1604 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:970U21 sense siNA AAGCAGUGGACACCAGGAGTT 1605 1191 AGAAGGGUACCUUGAACACUGGG 1292 IL13:1193U21 sense siNA AAGGGUACCUUGAACACUGTT 1606 391 CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:411L21 antisense siNA GCAGGUCCUUUACAAACUGTT 1607 (393C) 797 CACUUCACACACAGGCAACUGAG 1286 IL13:817L21 antisense siNA CAGUUGCCUGUGUGUGAAGTT 1608 (799C) 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:852L21 antisense siNA ACCAAGAAGAAGUGUGCCUTT 1609 (834C) 911 AAGACUGUGGCUGCUAGCACUUG 1288 IL13:931L21 antisense siNA AGUGCUAGCAGCCACAGUCTT 1610 (913C) 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:983L21 antisense siNA GGUGUCCACUGCUUUAGUGTT 1611 (965C) 965 CACUAAAGCAGUGGACACCAGGA 1290 IL13:985L21 antisense siNA CUGGUGUCCACUGCUUUAGTT 1612 (967C) 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:988L21 antisense siNA CUCCUGGUGUCCACUGCUUTT 1613 (970C) 1191 AGAAGGGUACCUUGAACACUGGG 1292 IL13:121IL21 antisense siNA CAGUGUUCAAGGUACCCUUTT 1614 (1193C) 391 CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:393U21 sense siNA B cAGuuuGuAAAGGAccuGcTT B 1615 stab04 797 CACUUCACACACAGGCAACUGAG 1286 IL13:799U21 sense siNA B cuucAcAcAcAGGcAAcuGTT B 1616 stab04 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:834U21 sense siNA B AGGcAcAcuucuucuuGGuTT B 1617 stab04 911 AAGACUGUGGCUGCUAGCACUUG 1288 IL13:913U21 sense siNA B GAcuGuGGcuGcuAGcAcuTT B 1618 stab04 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:965U21 sense siNA B cAcuAAAGcAGuGGAcAccTT B 1619 stab04 965 CACUAAAGCAGUGGACACCAGGA 1290 IL13:967U21 sense siNA B cuAAAGcAGuGGAcAccAGTT B 1620 stab04 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:970U21 sense siNA B AAGcAGuGGAcAccAGGAGTT B 1621 stab04 1191 AGAAGGGUACCUUGAACACUGGG 1292 IL13:1193U21 sense siNA B AAGGGuAccuuGAAcAcuGTT B 1622 stab04 391 CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:411L21 antisense siNA GcAGGuccuuuAcAAAcuGTsT 1623 (393C) stab05 797 CACUUCACACACAGGCAACUGAG 1286 IL13:817L21 antisense siNA cAGuuGccuGuGuGuGAAGTsT 1624 (799C) stab05 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:852L21 antisense siNA AccAAGAAGAAGuGuGccuTsT 1625 (834C) stab05 911 AAGACUGUGGCUGCUAGCACUUG 1288 IL13:931L21 antisense siNA AGuGcuAGcAGccAcAGucTsT 1626 (913C) stab05 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:983L21 antisense siNA GGuGuccAcuGcuuuAGuGTsT 1627 (965C) stab05 965 CACUAAAGCAGUGGACACCAGGA 1290 IL13:985L21 antisense siNA cuGGuGuccAcuGcuuuAGTsT 1628 (967C) stab05 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:988L21 antisense siNA cuccuGGuGuccAcuGcuuTsT 1629 (970C) stab05 1191 AGAAGGGUACCUUGAACACUGGG 1292 IL13:121IL21 antisense siNA cAGuGuucAAGGuAcccuuTsT 1630 (1193C) stab05 864 UAUUGUGUGUUAUUUAAAUGAGU 1293 33355 IL13:864U21 sense siNA B uuGuGuGuuAuuuAAAuGATT B 1631 stab07 865 AUUGUGUGUUAUUUAAAUGAGUG 1294 33356 IL13:865U21 sense siNA B uGuGuGuuAuuuAAAuGAGTT B 1632 stab07 866 UUGUGUGUUAUUUAAAUGAGUGU 1295 33357 IL13:866U21 sense siNA B GuGuGuuAuuuAAAuGAGuTT B 1633 stab07 863 UUAUUGUGUGUUAUUUAAAUGAG 1296 33358 IL13:863U21 sense siNA B AuuGuGuGuuAuuuAAAuGTT B 1634 stab07 200 UGCAAUGGCAGCAUGGUAUGGAG 1297 33359 IL13:200U21 sense siNA B cAAuGGcAGcAuGGuAuGGTT B 1635 stab07 201 GCAAUGGCAGCAUGGUAUGGAGC 1298 33360 IL13:201U21 sense siNA B AAuGGcAGcAuGGuAuGGATT B 1636 stab07 202 CAAUGGCAGCAUGGUAUGGAGCA 1299 33361 IL13:202U21 sense siNA B AuGGcAGcAuGGuAuGGAGTT B 1637 stab07 860 UUAUUAUUGUGUGUUAUUUAAAU 1300 33362 IL13:860U21 sense siNA B AuuAuuGuGuGuuAuuuAATT B 1638 stab07 861 UAUUAUUGUGUGUUAUUUAAAUG 1301 33363 IL13:861U21 sense siNA B uuAuuGuGuGuuAuuuAAA TT B 1639 stab07 862 AUUAUUGUGUGUUAUUUAAAUGA 1302 33364 IL13:862U21 sense siNA B uAuuGuGuGuuAuuuAAAuTT B 1640 stab07 391 CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:393U21 sense siNA B cAGuuuGuAAAGGAccuGcTT B 1641 stab07 797 CACUUCACACACAGGCAACUGAG 1286 IL13:799U21 sense siNA B cuucAcAcAcAGGcAAcuGTT B 1642 stab07 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:834U21 sense siNA B AGGcAcAcuucuucuuGGuTT B 1643 stab07 911 AAGACUGUGGCUGCUAGCACUUG 1288 IL13:913U21 sense siNA B GAcuGuGGcuGcuAGcAcuTT B 1644 stab07 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:965U21 sense siNA B cAcuAAAGcAGuGGAcAccTT B 1645 stab07 965 CACUAAAGCAGUGGACACCAGGA 1290 IL13:967U21 sense siNA B cuAAAGcAGuGGAcAccAGTT B 1646 stab07 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:970U21 sense siNA B AAGcAGuGGAcAccAGGAGTT B 1647 stab07 1191 AGAAGGGUACCUUGAACACUGGG 1292 IL13:1193U21 sense siNA B AAGGGuAccuuGAAcAcuGTT B 1648 stab07 391 CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:411L21 antisense siNA GcAGGuccuuuAcAAAcuGTsT 1649 (393C) stab11 797 CACUUCACACACAGGCAACUGAG 1286 IL13:817L21 antisense siNA cAGuuGccuGuGuGuGAAGTsT 1650 (799C) stab11 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:852L21 antisense siNA AccAAGAAGAAGuGuGccuTsT 1651 (834C) stab11 911 AAGACUGUGGCUGCUAGCACUUG 1288 IL13:931L21 antisense siNA AGuGcuAGcAGccAcAGucTsT 1652 (913C) stab11 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:983L21 antisense siNA GGuGuccAcuGcuuuAGuGTsT 1653 (965C) stab11 965 CACUAAAGCAGUGGACACCAGGA 1290 IL13:985L21 antisense siNA cuGGuGuccAcuGcuuuAGTsT 1654 (967C) stab11 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:988L21 antisense siNA cuccuGGuGuccAcuGcuuTsT 1655 (970C) stab11 1191 AGAAGGGUACCUUGAACACUGGG 1292 IL13:121IL21 antisense siNA cAGuGuucAAGGuAcccuuTsT 1656 (1193C) stab11 391 CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:393U21 sense siNA B cAGuuuGuAAAGGAccuGcTT B 1657 stab18 797 CACUUCACACACAGGCAACUGAG 1286 IL13:799U21 sense siNA B cuucAcAcAcAGGcAAcuGTT B 1658 stab18 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:834U21 sense siNA B AGGcAcAcuucuucuuGGuTT B 1659 stab18 911 AAGACUGUGGCUGCUAGCACUUG 1288 IL13:913U21 sense siNA B GAcuGuGGcuGcuAGcAcuTT B 1660 stab18 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:965U21 sense siNA B cAcuAAAGcAGuGGAcAccTT B 1661 stab18 965 CACUAAAGCAGUGGACACCAGGA 1290 IL13:967U21 sense siNA B cuAAAGcAGuGGAcAccAGTT B 1662 stab18 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:970U21 sense siNA B AAGcAGuGGAcAccAGGAGTT B 1663 stab18 1191 AGAAGGGUACCUUGAACACUGGG 1292 IL13:1193U21 sense siNA B AAGGGuAccuuGAAcAcuGTT B 1664 stab18 864 UAUUGUGUGUUAUUUAAAUGAGU 1293 33375 IL13:882L21 antisense siNA ucAuuuAAAuAAcAcAcAATsT 1665 (864C) stab08 865 AUUGUGUGUUAUUUAAAUGAGUG 1294 33376 IL13:883L21 antisense siNA cucAuuuAAAuAAcAcAcATsT 1666 (865C) stab08 866 UUGUGUGUUAUUUAAAUGAGUGU 1295 33377 IL13:884L21 antisense siNA AcucAuuuAAAuAAcAcAcTsT 1667 (866C) stab08 863 UUAUUGUGUGUUAUUUAAAUGAG 1296 33378 IL13:88IL21 antisense siNA cAuuuAAAuAAcAcAcAAuTsT 1668 (863C) stab08 200 UGCAAUGGCAGCAUGGUAUGGAG 1297 33379 IL13:218L21 antisense siNA ccAuAccAuGcuGccAuuGTsT 1669 (200C) stab08 201 GCAAUGGCAGCAUGGUAUGGAGC 1298 33380 IL13:219L21 antisense siNA uccAuAccAuGcuGccAuuTsT 1670 (201C) stab08 202 CAAUGGCAGCAUGGUAUGGAGCA 1299 33381 IL13:220L21 antisense siNA cuccAuAccAuGcuGccAuTsT 1671 (202C) stab08 860 UUAUUAUUGUGUGUUAUUUAAAU 1300 33382 IL13:878L21 antisense siNA uuAAAuAAcAcAcAAuAAuTsT 1672 (860C) stab08 861 UAUUAUUGUGUGUUAUUUAAAUG 1301 33383 IL13:879L21 antisense siNA uuuAAAuAAcAcAcAAuAATsT 1673 (861C) stab08 862 AUUAUUGUGUGUUAUUUAAAUGA 1302 33384 IL13:880L21 antisense siNA AuuuAAAuAAcAcAcAAuATsT 1674 (862C) stab08 391 CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:411L21 antisense siNA GcAGGuccuuuAcAAAcuGTsT 1675 (393C) stab08 797 CACUUCACACACAGGCAACUGAG 1286 IL13:817L21 antisense siNA cAGuuGccuGuGuGuGAAGTsT 1676 (799C) stab08 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:852L21 antisense siNA AccAAGAAGAAGuGuGccuTsT 1677 (834C) stab08 911 AAGACUGUGGCUGCUAGCACUUG 1288 IL13:931L21 antisense siNA AGuGcuAGcAGccAcAGucTsT 1678 (913C) stab08 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:983L21 antisense siNA GGuGuccAcuGcuuuAGuGTsT 1679 (965C) stab08 965 CACUAAAGCAGUGGACACCAGGA 1290 IL13:985L21 antisense siNA cuGGuGuccAcuGcuuuAGTsT 1680 (967C) stab08 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:988L21 antisense siNA cuccuGGuGuccAcuGcuuTsT 1681 (970C) stab08 1191 AGAAGGGUACCUUGAACACUGGG 1292 IL13:121IL21 antisense siNA cAGuGuucAAGGuAcccuuTsT 1682 (1193C) stab08 391 CCCAGUUUGUAAAGGACCUGCUC 1285 36890 IL13:393U21 sense siNA B CAGUUUGUAAAGGACCUGCTT B 1683 stab09 797 CACUUCACACACAGGCAACUGAG 1286 36891 IL13:799U21 sense siNA B CUUCACACACAGGCAACUGTT B 1684 stab09 832 UCAGGCACACUUCUUCUUGGUCU 1287 36892 IL13:834U21 sense siNA B AGGCACACUUCUUCUUGGUTT B 1685 stab09 911 AAGACUGUGGCUGCUAGCACUUG 1288 36893 IL13:913U21 sense siNA B GACUGUGGCUGCUAGCACUTT B 1686 stab09 963 AGCACUAAAGCAGUGGACACCAG 1289 36894 IL13:965U21 sense siNA B CACUAAAGCAGUGGACACCTT B 1687 stab09 965 CACUAAAGCAGUGGACACCAGGA 1290 36895 IL13:967U21 sense siNA B CUAAAGCAGUGGACACCAGTT B 1688 stab09 968 UAAAGCAGUGGACACCAGGAGUC 1291 36896 IL13:970U21 sense siNA B AAGCAGUGGACACCAGGAGTT B 1689 stab09 1191 AGAAGGGUACCUUGAACACUGGG 1292 36897 IL13:1193U21 sense siNA B AAGGGUACCUUGAACACUGTT B 1690 stab09 391 CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:411L21 antisense siNA GCAGGUCCUUUACAAACUGTsT 1691 (393C) stab10 797 CACUUCACACACAGGCAACUGAG 1286 IL13:817L21 antisense siNA CAGUUGCCUGUGUGUGAAGTsT 1692 (799C) stab10 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:852L21 antisense siNA ACCAAGAAGAAGUGUGCCUTsT 1693 (834C) stab10 911 AAGACUGUGGCUGCUAGCACUUG 1288 IL13:931L21 antisense siNA AGUGCUAGCAGCCACAGUCTsT 1694 (913C) stab10 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:983L21 antisense siNA GGUGUCCACUGCUUUAGUGTsT 1695 (965C) stab10 965 CACUAAAGCAGUGGACACCAGGA 1290 IL13:985L21 antisense siNA CUGGUGUCCACUGCUUUAGTsT 1696 (967C) stab10 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:988L21 antisense siNA CUCCUGGUGUCCACUGCUUTsT 1697 (970C) stab10 1191 AGAAGGGUACCUUGAACACUGGG 1292 IL13:121IL21 antisense siNA CCAGUGUUCAAGGUACCCUUTsT 1698 (1193C) stab10 391 CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:411L21 antisense siNA GcAGGuccuuuAcAAAcuGTT B 1699 (393C) stab19 797 CACUUCACACACAGGCAACUGAG 1286 IL13:817L21 antisense siNA cAGuuGccuGuGuGuGAAGTT B 1700 (799C) stab19 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:852L21 antisense siNA AccAAGAAGAAGuGuGccuTT B 1701 (834C) stab19 911 AAGACUGUGGCUGCUAGCACUUG 1288 IL13:931L21 antisense siNA AGuGcuAGcAGccAcAGucTT B 1702 (913C) stab19 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:983L21 antisense siNA GGuGuccAcuGcuuuAGuGTT B 1703 (965C) stab19 965 CACUAAAGCAGUGGACACCAGGA 1290 IL13:985L21 antisense siNA cuGGuGuccAcuGcuuuAGTT B 1704 (967C) stab19 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:988L21 antisense siNA cuccuGGuGuccAcuGcuuTT B 1705 (970C) stab19 1191 AGAAGGGUACCUUGAACACUGGG 1292 IL13:121IL21 antisense siNA cAGuGuucAAGGuAcccuuTT B 1706 (1193C) stab19 391 CCCAGUUUGUAAAGGACCUGCUC 1285 36898 IL13:411L21 antisense siNA GCAGGUCCUUUACAAACUGTT B 1707 (393C) stab22 797 CACUUCACACACAGGCAACUGAG 1286 36899 IL13:817L21 antisense siNA CAGUUGCCUGUGUGUGAAGTT B 1708 (799C) stab22 832 UCAGGCACACUUCUUCUUGGUCU 1287 36900 IL13:852L21 antisense siNA ACCAAGAAGAAGUGUGCCUTT B 1709 (834C) stab22 911 AAGACUGUGGCUGCUAGCACUUG 1288 36901 IL13:931L21 antisense siNA AGUGCUAGCAGCCACAGUCTTB 1710 (913C) stab22 963 AGCACUAAAGCAGUGGACACCAG 1289 36902 IL13:983L21 antisense siNA GGUGUCCACUGCUUUAGUGTT B 1711 (965C) stab22 965 CACUAAAGCAGUGGACACCAGGA 1290 36903 IL13:985L21 antisense siNA CUGGUGUCCACUGCUUUAGTT B 1712 (967C) stab22 968 UAAAGCAGUGGACACCAGGAGUC 1291 36904 IL13:988L21 antisense siNA CUCCUGGUGUCCACUGCUUTT B 1713 (970C) stab22 1191 AGAAGGGUACCUUGAACACUGGG 1292 36905 IL13:121IL21 antisense siNA CAGUGUUCAAGGUACCCUUTT B 1714 (1193C) stab22 IL13R 408 AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:410U21 sense siNA GGUGAUCCUGAGUCUGCUGTT 1715 657 UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:659U21 sense siNA GUCAAGGAUAAUGCAGGAATT 1716 871 CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:873U21 sense siNA UCCAAGAGGCUAAAUGUGATT 1717 1276 GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1278U21 sense siNA AAACCGACUCUGUAGUGCUTT 1718 1308 UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1310U21 sense siNA AAGAAAGCCUCUCAGUGAUTT 1719 1424 ACUGCACCAUUUAAAAACAGGCA 1308 IL13RA1:1426U21 sense siNA UGCACCAUUUAAAAACAGGTT 1720 2186 CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2188U21 sense siNA GCAUUUUCCUCUGCUUUGATT 1721 2270 CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2272U21 sense siNA AAGACCUUUCAAAGCCAUUTT 1722 408 AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:428L21 antisense CAGCAGACUCAGGAUCACCTT 1723 siNA (410C) 657 UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:677L21 antisense UUCCUGCAUUAUCCUUGACTT 1724 siNA (659C) 871 CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:89IL21 antisense UCACAUUUAGCCUCUUGGATT 1725 siNA (873C) 1276 GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1296L21 antisense AGCACUACAGAGUCGGUUUTT 1726 siNA (1278C) 1308 UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1328L21 antisense AUCACUGAGAGGCUUUCUUTT 1727 siNA (1310C) 1424 ACUGCACCAUUUAAAAACAGGCA 1308 IL13RA1:1444L21 antisense CCUGUUUUUAAAUGGUGCATT 1728 siNA (1426C) 2186 CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2206L21 antisense UCAAAGCAGAGGAAAAUGCTT 1729 siNA (2188C) 2270 CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2290L21 antisense AAUGGCUUUGAAAGGUCUUTT 1730 siNA (2272C) 408 AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:410U21 sense siNA B GGuGAuccuGAGucuGcuGTT B 1731 stab04 657 UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:659U21 sense siNA B GucAAGGAuAAuGcAGGAATT B 1732 stab04 871 CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:873U21 sense siNA B uccAAGAGGcuAAAuGuGATT B 1733 stab04 1276 GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1278U21 sense siNA B AAAccGAcucuGuAGuGcuTT B 1734 stab04 1308 UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1310U21 sense siNA B AAGAAAGccucucAGuGAuTT B 1735 stab04 1424 ACUGCACCAUUUAAAAACAGGCA 1308 IL13RA1:1426U21 sense siNA B uGcAccAuuuAAAAAcAGGTT B 1736 stab04 2186 CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2188U21 sense siNA B GcAuuuuccucuGcuuuGATT B 1737 stab04 2270 CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2272U21 sense siNA B AAGAccuuucAAAGccAuuTT B 1738 stab04 408 AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:428L21 antisense cAGcAGAcucAGGAucAccTsT 1739 siNA (410C) stab05 657 UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:677L21 antisense uuccuGcAuuAuccuuGAcTsT 1740 siNA (659C) stab05 871 CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:891L21 antisense ucAcAuuuAGccucuuGGATsT 1741 siNA (873C) stab05 1276 GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1296L21 antisense AGcAcuAcAGAGucGGuuuTsT 1742 siNA (1278C) stab05 1308 UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1328L21 antisense AucAcuGAGAGGcuuucuuTsT 1743 siNA (1310C) stab05 1424 ACUGCACCAUUUAAAAACAGGCA 1308 IL13RA1:1444L21 antisense ccuGuuuuuAAAuGGuGcATsT 1744 siNA (1426C) stab05 2186 CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2206L21 antisense ucAAAGcAGAGGAAAAuGcTsT 1745 siNA (2188C) stab05 2270 CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2290L21 antisense AAuGGcuuuGAAAGGucuuTsT 1746 siNA (2272C) stab05 408 AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:410U21 sense siNA B GGuGAuccuGAGucuGcuGTT B 1747 stab07 657 UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:659U21 sense siNA B GucAAGGAuAAuGcAGGAATT B 1748 stab07 871 CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:873U21 sense siNA B uccAAGAGGcuAAAuGuGATT B 1749 stab07 1276 GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1278U21 sense siNA B AAAccGAcucuGuAGuGcuTT B 1750 stab07 1308 UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1310U21 sense siNA B AAGAAAGccucucAGuGAuTT B 1751 stab07 1424 ACUGCACCAUUUAAAAACAGGCA 1308 IL13RA1:1426U21 sense siNA B uGcAccAuuuAAAAAcAGGTT B 1752 stab07 2186 CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2188U21 sense siNA B GcAuuuuccucuGcuuuGATT B 1753 stab07 2270 CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2272U21 sense siNA B AAGAccuuucAAAGccAuuTT B 1754 stab07 408 AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:428L21 antisense cAGcAGAcucAGGAucAccTsT 1755 siNA (410C) stab11 657 UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:677L21 antisense uuccuGcAuuAuccuuGAcTsT 1756 siNA (659C) stab11 871 CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:891L21 antisense ucAcAuuuAGccucuuGGATsT 1757 siNA (873C) stab11 1276 GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1296L21 antisense AGcAcuAcAGAGucGGuuuTsT 1758 siNA (1278C) stab11 1308 UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1328L21 antisense AucAcuGAGAGGcuuucuuTsT 1759 siNA (1310C) stab11 1424 ACUGCACCAUUUAAAAACAGGCA 1308 IL13RA1:1444L21 antisense ccuGuuuuuAAAuGGuGcATsT 1760 siNA (1426C) stab11 2186 CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2206L21 antisense ucAAAGcAGAGGAAAAuGcTsT 1761 siNA (2188C) stab11 2270 CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2290L21 antisense AAuGGcuuuGAAAGGucuuTsT 1762 siNA (2272C) stab11 408 AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:410U21 sense siNA B GGuGAuccuGAGucuGcuGTT B 1763 stab18 657 UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:659U21 sense siNA B GucAAGGAuAAuGcAGGAATT B 1764 stab18 871 CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:873U21 sense siNA B uccAAGAGGcuAAAuGuGATT B 1765 stab18 1276 GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1278U21 sense siNA B AAAccGAcucuGuAGuGcuTT B 1766 stab18 1308 UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1310U21 sense siNA B AAGAAAGccucucAGuGAuTT B 1767 stab18 1424 ACUGCACCAUUUAAAAACAGGCA 1308 ILl3RAl:1426U21 sense siNA B uGcAccAuuuAAAAAcAGGTT B 1768 stab18 2186 CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2188U21 sense siNA B GcAuuuuccucuGcuuuGATT B 1769 stab18 2270 CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2272U21 sense siNA B AAGAccuuucAAAGccAuuTT B 1770 stab18 408 AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:428L21 antisense cAGcAGAcucAGGAucAccTsT 1771 siNA (410C) stab08 657 UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:677L21 antisense uuccuGcAuuAuccuuGAcTsT 1772 siNA (659C) stab08 871 0GU00AAGAGG0UAAAUGUGAGA 1305 L13RA1:891L21 antisense ucAcAuuuAGccucuuGGATsT 1773 siNA (873C) stab08 1276 GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1296L21 antisense AGcAcuAcAGAGucGGuuuTsT 1774 siNA (1278C) stab08 1308 UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1328L21 antisense AucAcuGAGAGGcuuucuuTsT 1775 siNA (1310C) stab08 1424 ACUGCACCAUUUAAAAACAGGCA 1308 IL13RA1:1444L21 antisense ccuGuuuuuAAAuGGuGcATsT 1776 siNA (1426C) stab08 2186 CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2206L21 antisense ucAAAGcAGAGGAAAAuGcTsT 1777 siNA (2188C) stab08 2270 CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2290L21 antisense AAuGGcuuuGAAAGGucuuTsT 1778 siNA (2272C) stab08 408 AAGGUGAUCCUGAGUCUGCUGUG 1303 36906 IL13RA1:410U21 sense siNA B GGUGAUCCUGAGUCUGCUGTT B 1779 stab09 657 UGGUCAAGGAUAAUGCAGGAAAA 1304 36907 IL13RA1:659U21 sense siNA B GUCAAGGAUAAUGCAGGAATT B 1780 stab09 871 CGUCCAAGAGGCUAAAUGUGAGA 1305 36908 IL13RA1:873U21 sense siNA B UCCAAGAGGCUAAAUGUGATT B 1781 stab09 1276 GGAAACCGACUCUGUAGUGCUGA 1306 36909 IL13RA1:1278U21 sense siNA B AAACCGACUCUGUAGUGCUTT B 1782 stab09 1308 UGAAGAAAGCCUCUCAGUGAUGG 1307 36910 IL13RA1:1310U21 sense siNA B AAGAAAGCCUCUCAGUGAUTT B 1783 stab09 1424 ACUGCACCAUUUAAAAACAGGCA 1308 36911 IL13RA1:1426U21 sense siNA B UGCACCAUUUAAAAACAGGTT B 1784 stab09 2186 CAGCAUUUUCCUCUGCUUUGAAA 1309 36912 IL13RA1:2188U21 sense siNA B GCAUUUUCCUCUGCUUUGATT B 1785 stab09 2270 CCAAGACCUUUCAAAGCCAUUUU 1310 36913 IL13RA1:2272U21 sense siNA B AAGACCUUUCAAAGCCAUUTT B 1786 stab09 408 AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:428L21 antisense CAGCAGACUCAGGAUCACCTsT 1787 siNA (410C) stab10 657 UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:677L21 antisense UUCCUGCAUUAUCCUUGACTsT 1788 siNA (659C) stab10 871 CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:891L21 antisense UCACAUUUAGCCUCUUGGATsT 1789 siNA (873C) stab10 1276 GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1296L21 antisense AGCACUACAGAGUCGGUUUTsT 1790 siNA (1278C) stab10 1308 UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1328L21 antisense AUCACUGAGAGGCUUUCUUTsT 1791 siNA (1310C) stab10 1424 ACUGCACCAUUUAAAAACAGGCA 1308 IL13RA1:1444L21 antisense CCUGUUUUUAAAUGGUGCATsT 1792 siNA (1426C) stab10 2186 CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2206L21 antisense UCAAAGCAGAGGAAAAUGCTsT 1793 siNA (2188C) stab10 2270 CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2290L21 antisense AAUGGCUUUGAAAGGUCUUTsT 1794 siNA (2272C) stab10 408 AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:428L21 antisense cAGcAGAcucAGGAucAccTT B 1795 siNA (410C) stab19 657 UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:677L21 antisense uuccuGcAuuAuccuuGAcTT B 1796 siNA (659C) stab19 871 CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:891L21 antisense ucAcAuuuAGccucuuGGATT B 1797 siNA (873C) stab19 1276 GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1296L21 antisense AGcAcuAcAGAGucGGuuuTT B 1798 siNA (1278C) stab19 1308 UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1328L21 antisense AucAcuGAGAGGcuuucuuTT B 1799 siNA (1310C) stab19 1424 ACUGCACCAUUUAAAAACAGGCA 1308 IL13RA1:1444L21 antisense ccuGuuuuuAAAuGGuGcATT B 1800 siNA (1426C) stab19 2186 CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2206L21 antisense ucAAAGcAGAGGAAAAuGcTT B 1801 siNA (2188C) stab19 2270 CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2290L21 antisense AAuGGcuuuGAAAGGucuuTT B 1802 siNA (2272C) stab19 408 AAGGUGAUCCUGAGUCUGCUGUG 1303 36914 IL13RA1:428L21 antisense CAGCAGACUCAGGAUCACCTT B 1803 siNA (410C) stab22 657 UGGUCAAGGAUAAUGCAGGAAAA 1304 36915 IL13RA1:677L21 antisense UUCCUGCAUUAUCCUUGACTT B 1804 siNA (659C) stab22 871 CGUCCAAGAGGCUAAAUGUGAGA 1305 36916 IL13RA1:891L21 antisense UCACAUUUAGCCUCUUGGATT B 1805 siNA (873C) stab22 1276 GGAAACCGACUCUGUAGUGCUGA 1306 36917 IL13RA1:1296L21 antisense AGCACUACAGAGUCGGUUUTT B 1806 siNA (1278C) stab22 1308 UGAAGAAAGCCUCUCAGUGAUGG 1307 36918 IL13RA1:1328L21 antisense AUCACUGAGAGGCUUUCUUTT B 1807 siNA (1310C) stab22 1424 ACUGCACCAUUUAAAAACAGGCA 1308 36919 IL13RA1:1444L21 antisense CCUGUUUUUAAAUGGUGCATT B 1808 siNA (1426C) stab22 2186 CAGCAUUUUCCUCUGCUUUGAAA 1309 36920 IL13RA1:2206L21 antisense UCAAAGCAGAGGAAAAUGCTT B 1809 siNA (2188C) stab22 2270 CCAAGACCUUUCAAAGCCAUUUU 1310 36921 IL13RA1:2290L21 antisense AAUGGCUUUGAAAGGUCUUTT B 1810 siNA (2272C) stab22 Uppercase = ribonucleotide u, c = 2′-deoxy-2′-fluoro U, C T = thymidine B = inverted deoxy abasic s = phosphorothioate linkage A = deoxy Adenosine G = deoxy Guanosine G = 2′-O-methyl Guanosine A = 2′-O-methyl Adenosine

TABLE IV Non-limiting examples of Stabilization Chemistries for chemically modified siNA constructs Chemistry pyrimidine Purine cap p = S Strand “Stab 00” Ribo Ribo TT at S/AS 3′-ends “Stab 1” Ribo Ribo — 5 at 5′-end S/AS 1 at 3′-end “Stab 2” Ribo Ribo — All Usually AS linkages “Stab 3” 2′-fluoro Ribo — 4 at 5′-end Usually S 4 at 3′-end “Stab 4” 2′-fluoro Ribo 5′ and — Usually S 3′-ends “Stab 5” 2′-fluoro Ribo — 1 at 3′-end Usually AS “Stab 6” 2′-O-Methyl Ribo 5′ and — Usually S 3′-ends “Stab 7” 2′-fluoro 2′-deoxy 5′ and — Usually S 3′-ends “Stab 8” 2′-fluoro 2′-O- — 1 at 3′-end Usually AS Methyl “Stab 9” Ribo Ribo 5′ and — Usually S 3′-ends “Stab 10” Ribo Ribo — 1 at 3′-end Usually AS “Stab 11” 2′-fluoro 2′-deoxy — 1 at 3′-end Usually AS “Stab 12” 2′-fluoro LNA 5′ and Usually S 3′-ends “Stab 13” 2′-fluoro LNA 1 at 3′-end Usually AS “Stab 14” 2′-fluoro 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab 15” 2′-deoxy 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab 16” Ribo 2′-O- 5′ and Usually S Methyl 3′-ends “Stab 17” 2′-O-Methyl 2′-O- 5′ and Usually S Methyl 3′-ends “Stab 18” 2′-fluoro 2′-O- 5′ and 1 at 3′-end Usually S Methyl 3′-ends “Stab 19” 2′-fluoro 2′-O- 3′-end Usually AS Methyl “Stab 20” 2′-fluoro 2′-deoxy 3′-end Usually AS “Stab 21” 2′-fluoro Ribo 3′-end Usually AS “Stab 22” Ribo Ribo 3′-end - Usually AS “Stab 23” 2′-fluoro* 2′-deoxy* 5′ and Usually S 3′-ends “Stab 24” 2′-fluoro* 2′-O- — 1 at 3′-end Usually AS Methyl* “Stab 25” 2′-fluoro* 2′-O- — 1 at 3′-end Usually AS Methyl* CAP = any terminal cap, see for example FIG. 10. All Stab 1-25 chemistries can comprise 3′-terminal thymidine (TT) residues All Stab 1-25 chemistries typically comprise about 21 nucleotides, but can vary as described herein. S = sense strand AS = antisense strand *Stab 23 has single ribonucleotide adjacent to 3′-CAP *Stab 24 has single ribonucleotide at 5′-terminus *Stab 25 has three ribonucleotides at 5′-terminus

TABLE V Reagent Equivalents Amount Wait Time* DNA Wait Time* 2′-O-methyl Wait Time* RNA A. 2.5 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole 23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL 5 sec 5 sec 5 sec N-Methyl 186 233 μL 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL 21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage 12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B. 0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 μL 45 sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min 465 sec Acetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl 1245 124 μL 5 sec 5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 sec Iodine 20.6 244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300 sec 300 sec Acetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle 96 well Instrument Equivalents: DNA/ Amount: DNA/2′-O- Wait Time* 2′-O- Reagent 2′-O-methyl/Ribo methyl/Ribo Wait Time* DNA methyl Wait Time* Ribo Phosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 sec S-Ethyl Tetrazole 70/105/210 40/60/120 μL 60 sec 180 min 360 sec Acetic Anhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl 502/502/502 50/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA 238/475/475 250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30 sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 sec Acetonitrile NA 1150/1150/1150 μL NA NA NA Wait time does not include contact time during delivery. Tandem synthesis utilizes double coupling of linker molecule 

1. A chemically modified nucleic acid molecule, wherein: (a) the nucleic acid molecule comprises a sense strand and a separate antisense strand, each strand having one or more pyrimidine nucleotides and one or more purine nucleotides; (b) each strand of the nucleic acid molecule is independently 18 to 27 nucleotides in length; (c) an 18 to 27 nucleotide sequence of the antisense strand is complementary to a human interleukin-4 receptor (IL-4R) comprising SEQ ID NO: 1833; (d) an 18 to 27 nucleotide sequence of the sense strand is complementary to the antisense strand and comprises an 18 to 27 nucleotide sequence of the human RNA sequence; and (e) 50 percent or more of the nucleotides in each strand comprise a 2′-sugar modification, wherein the 2′-sugar modification of any of the pyrimidine nucleotides differs from the 2′-sugar modification of any of the purine nucleotides.
 2. The nucleic acid molecule of claim 1, wherein the 2′-sugar modification of any of the purine nucleotides in the sense strand differs from the 2′-sugar modification of any of the purine nucleotides in the antisense strand.
 3. The nucleic acid molecule of claim 1, wherein the 2′-sugar modification is selected from the group consisting of 2′-deoxy-2′-fluoro, 2′-O-methyl, and 2′-deoxy.
 4. The nucleic acid of claim 3, wherein the 2′-deoxy-2′-fluoro sugar modification is a pyrimidine modification.
 5. The nucleic acid of claim 3, wherein the 2′-deoxy sugar modification is a pyrimidine modification.
 6. The nucleic acid of claim 3, wherein the 2′-O-methyl sugar modification is a pyrimidine modification.
 7. The nucleic acid molecule of claim 4, wherein said pyrimidine modification is in the sense strand, the antisense strand, or both the sense strand and antisense strand.
 8. The nucleic acid molecule of claim 6, wherein said pyrimidine modification is in the sense strand, the antisense strand, or both the sense strand and antisense strand.
 9. The nucleic acid molecule of claim 3, wherein the 2′-deoxy sugar modification is a purine modification.
 10. The nucleic acid molecule of claim 3, wherein the 2′-O-methyl sugar modification is a purine modification.
 11. The nucleic acid molecule of claim 9, wherein the purine modification is in the sense strand.
 12. The nucleic acid molecule of claim 10, wherein the purine modification is in the antisense strand.
 13. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises ribonucleotides.
 14. The nucleic acid molecule of claim 1, wherein the sense strand includes a terminal cap moiety at the 5′-end, the 3′-end, or both of the 5′- and 3′-ends.
 15. The nucleic acid molecule of claim 14, wherein the terminal cap moiety is an inverted deoxy abasic moiety.
 16. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule includes one or more phosphorothioate internucleotide linkages.
 17. The nucleic acid molecule of claim 16, wherein one of the phosphorothioate internucleotide linkages is at the 3′-end of the antisense strand.
 18. The nucleic acid molecule of claim 1, wherein the 5′-end of the antisense strand includes a terminal phosphate group.
 19. The nucleic acid molecule of claim 1, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand include a 3′-overhang.
 20. A composition comprising the nucleic acid molecule of claim 1, in a pharmaceutically acceptable carrier or diluent. 